Since the classic studies of Pavlov, a wide variety of researchers have investigated the process of classical conditioning and used it to investigate animal behaviour. Recently, researchers in animal behaviour have discovered important new benefits associated with classical conditioning, now being applied as a way of enhancing well-being . Pavlovian conditioning occurs when an association between two stimuli, a neutral stimulus and a biologically relevant stimulus, is created by repeated paired presentations of both stimuli. When the presentation of the neutral stimulus is followed by a delay, before the biological stimulus is presented, expectation or anticipatory behaviour is generated. Anticipatory behaviour is known to elicit the release of dopamine. As play behaviour is also known to result in the release of dopamine, and correlates with positive welfare state, studies of the relationship between anticipatory behaviour and play may generate important new insights for animal welfare. In this study I assessed if (i) early weaning influenced the development of playful attacks and pinning behaviour in rats and (ii) the effects of a Pavlovian conditioning paradigm on the occurrence of play in both early - and normally - weaned rats. Male pups were assigned to one of the following treatment groups: (1) an early weaned (EW) group without exposure to conditioning, (2) a normally weaned (NW) group without exposure to conditioning, (3) an EW group with a conditional stimulus (CS) paired to an unconditional stimulus (US) to generate anticipatory behaviour (CS-US), (4) an EW group with CS-US unpaired, (5) an EW group with US only and (6) a NW group with US only. Animals in each group were observed for one hour each week from 4 to 7 weeks of life to record the frequency of playful attacks and pinning behaviour. EW pups that were not exposed to any form of conditioning had a significant reduction in the frequency of playful attacks relative to the NW pups without conditioning; pinning frequencies were low, but the effect was not statistically significant. The difference in frequencies of playful attacks between the NW group without exposure to conditioning and all the EW groups was statistically significant, with the NW groups displaying the highest frequencies of playful behaviour; pinning changes across those same treatment groups were not significant. Interestingly, the EW group under Pavlovian conditioning had a higher mean frequency of playful attacks and pinning behaviour relative to the EW control groups (i.e. EW with CS-US unpaired and EW with US only). Eliciting anticipatory behaviour may explain the increased levels of play behaviour in the EW conditioned group. Weight increases were similar across all EW and NW groups. The results of this study are discussed in relation to the effects that early weaning has on behavioural development, the potential benefits of using anticipatory behaviour to reduce the impacts of stressful events, and its positive effects in the development of play behaviour in rats. The findings support the idea that a Pavlovian conditioning paradigm can be used to enhance the welfare of animals and that the expectation for the reward has a greater effect than the reward itself.