PPCM (previously designated SAMMA) is a contraceptive microbicide in preclinical development. Its contraceptive activity is due in part to its ability to promote premature acrosomal loss. Prior studies showed that PPCM-induced human acrosomal loss (PAL) is Ca²⁺-dependent (Anderson et al, 2006). This study was carried out to determine transduction elements downstream from Ca²⁺ entry. PAL is inhibited by inhibitors selective for endothelial type nitric oxide synthase. PAL is completely inhibited by 0.1 µM ODQ (soluble guanylate cyclase inhibitor). PAL is inhibited by protein kinase G inhibitors with selectivity for the type-II isotype. Several inhibitors of the nitric oxide/cGMP/protein kinase G pathway induce Ca²⁺-dependent acrosomal loss when added alone. These responses are inhibited by nifedipine, a blocker of Cav1.x voltage-dependent channels. Acrosomal loss induced by the nitric oxide donor, SNAP (SNAL), does not require added Ca²⁺. Sperm production of nitric oxide is increased by PPCM, an effect inhibited by nitro-L-arginine (nitric oxide synthase inhibitor). Although inhibited by ODQ, SNAL and acrosomal loss induced by other nitric oxide donors are unaffected by KT5823 (protein kinase G inhibitor). Unlike PAL, SNAL is partially inhibited by KT5720 (protein kinase A inhibitor) and genistein (protein tyrosine kinase inhibitor). Acrosomal loss response to PPCM and SNAP added in combination suggests that these agents act by independent mechanisms. A PPCM derivative was synthesized, in which a nitric oxide donor was esterified to PPCM (NOSPPA-23). NOSPPA-23 induces acrosomal loss with or without added Ca²⁺. The ED50 of NOSPPA-23 (4.8 nM) in the presence of Ca²⁺ is 35-fold less than that of PPCM. These findings suggest the following: 1) elements responsible for PAL include epithelial nitric oxide synthase, soluble guanylate cyclase and type II protein kinase G; 2) the resting state of the nitric oxide/cGMP/protein kinase G pathway is a determinant of acrosomal status; 3) PPCM and nitric oxide donors induce acrosomal loss via nitric oxide, but through independent pathways; and 4) covalent attachment of an nitric oxide donor to PPCM provides synergistic efficacy as a stimulus of acrosomal loss. Further studies with this novel prototype as an improved contraceptive microbicide are warranted.