Cooperation is — despite not being predicted by game theory — a widely documented aspect of human behaviour in Prisoner’s Dilemma (PD) situations. This article presents a comparison between subjects restricted to playing pure strategies and subjects allowed to play mixed strategies in a one-shot symmetric PD laboratory experiment. Subjects interact with 10 other subjects and take their decisions all at once. Because subjects in the mixed-strategy treatment group are allowed to condition their level of cooperation more precisely on their beliefs about their counterparts’ level of cooperation, we predicted the cooperation rate in the mixed-strategy treatment group to be higher than in the pure-strategy control group. The results of our experiment reject our prediction: even after controlling for beliefs about the other subjects’ level of cooperation, we find that cooperation in the mixed-strategy group is lower than in the pure-strategy group. We also find, however, that subjects in the mixedstrategy group condition their cooperative behaviour more closely on their beliefs than in the pure-strategy group. In the mixed-strategy group, most subjects choose intermediate levels of cooperation.