The fertilization in vitro of mouse with intact zonae pellucidae by mouse cauda epididymal spermatozoa was inhibited in a concentration- dependent fashion by 3-quinuclidinyl benzilate (QNB), normally used as a specific antagonist for the muscarinic class of cholinergic receptors. Inhibition was observed with both cumulus-intact and cumulus-free preparations. QNB at 50 microM inhibited fertilization of cumulus-free eggs by greater than 90% but had no effect on the fertilization of zona-free eggs. At this concentrations, QNB had no adverse effect on sperm motility, nor did it prevent binding of spermatozoa to the zona pellucida. The inhibitory effects of QNB were fully reversible. QNB is therefore a useful specific inhibitor of zona penetration. Spermatozoa in the in vitro fertilization medium bound QNB with a concentration dependence which matched that of the inhibition of fertilization. This binding was saturable and corresponded to 700 pmole/10(7) cells with KD = 10 microM. The in vitro fertilization medium contains 2% (w/v) bovine serum albumin (BSA) which also binds QNB according to the mass action law. The large amount of QNB bound to sperm in this medium appears to be QNB binding to BSA adsorbed on the sperm cell surface: these spermatozoa bind QNB specifically in the absence of BSA with a saturable capacity of only 70 fmole/10(7) cells with KD = 5 nM. Calculation of the distribution of QNB between BSA binding sites and sperm surface binding sites in the in vitro fertilization medium indicates that the specific sperm sites become saturated with the same concentration dependence as inhibition of fertilization. However, the dissociation rate of QNB from sperm in both the presence and absence of BSA is too rapid to permit confirmation of these sites as the locus of the inhibitory effect; this locus remains to be clarified.