The purpose of the present experiments was to demonstrate conditioning of anticipatory pupillary dilation responses in humans as previous efforts to condition pupillary dilation had yielded inconsistent results. In those studies, using an aversive unconditioned stimulus (US) had yielded more promising results than using non-aversive USs. In three experiments we utilized a reaction time task as a non-aversive US and observed reliable differential conditioning of anticipatory pupillary dilation. Differential responding was evident within very few trials and was largest late in the anticipatory interval. Experiment 4 replicated these findings using an aversive US (electric shock). In measuring anticipatory responses in a differential conditioning preparation all four experiments provided clear evidence for Pavlovian conditioning of pupillary dilation in humans.