Brain microdialysis was used to study changes in dopamine in the nucleus accumbens and the dorsal striatum during associative learning between two neutral stimuli, flashing light and tone, presented on a paired schedule during stage 1 of a sensory preconditioning paradigm. The tone was subsequently paired with mild footshock using standard aversive conditioning procedures and the formation of a conditioned association between the flashing light and the tone in stage 1 was assessed by measuring the ability of the flashing light to elicit the same conditioned response as the tone when presented at test. The first experiment used behavioural monitoring only, to establish stimulus parameters for subsequent microdialysis experiments. Animals receiving paired presentation of the light and tone in stage 1 showed a conditioned suppression of licking to the light as well as to the tone, indicating that associative learning between the flashing light and the tone had occurred during stage 1, whilst in a separate group of animals given the same stimuli over the same time period but on an explicitly non-paired schedule, the conditioned emotional response was seen to the tone, but not to the light, showing that no association had been formed between the two stimuli during stage 1. In dialysis experiments using the same procedure, we measured a two-fold rise in dopamine in the nucleus accumbens during paired presentation of flashing light and tone, but not during non-paired presentation of the two stimuli. On subsequent test presentation of the two stimuli, we saw increases in accumbal dopamine on presentation of the tone in both groups, reflecting the formation of an association with the footshock in both. However the flashing light elicited an increase in dopamine only in the group which had received paired presentation at stage 1. Thus accumbal dopamine release at test is correlated to the ability of the stimulus to evoke a conditioned response measured behaviourally. Hypotheses of the behavioural function of the mesolimbic dopamine system centre on its role in mediating the effects of biological reinforcers, both rewarding and aversive, conditioned and unconditioned. The present results, showing increases in extracellular dopamine in the nucleus accumbens when an association is formed between two stimuli of which neither is a biological reinforcer nor, prior to formation of the association, affects dopamine levels, suggest a role for accumbal dopamine in the modulation of associative learning in general, not only that involving reinforcement.