Cytochalasin B inhibited uptake of 2-deoxy-D-glucose in rat spermatids (Ki = 6.3 X 10(-7) M). This inhibition was reversible. The [3H]cytochalasin B was found to bind to spermatid plasma membranes at low drug concentrations. Lineweaver-Burk plots of binding data indicated that there was a class of high affinity sites (Kd = 2.9 X 10(-7) M). The results suggest that the high affinity cytochalasin B binding sites of spermatids are membrane-associated substances and are intimately related to the glucose transport system.