This study investigates the dynamics of serine/threonine (S/T) protein phosphorylation in sperm incubated under capacitating (C) conditions using the boar as a model system. For the first time, this approach has identified multiple dephosphorylation events that occur in a bicarbonate-dependent fashion. Different phospho-(S/T) kinase substrate antibodies were used, and dephosphorylation of 5 S/T phosphoproteins was observed in C sperm compared with noncapacitated (N) cells. Specifically, dephosphorylation of 96-, 90-, 64-, and 55-kd proteins was detected by immunoblotting using 2 phospho-Akt substrate antibodies and a phosphoprotein kinase A substrate antibody. In addition, dephosphorylation of a 105-kd protein was detected using a phospho-ATM/ATR substrate antibody. In contrast, no dephosphorylation was observed using a phosphoprotein kinase C substrate antibody, and increased tyrosine phosphorylation of 32- and 20-kd proteins was detected in C compared with N sperm. Immunolocalization experiments revealed subtle changes in the pattern expression as well as a reduction of phosphorylation in C sperm. Whereas sperm incubated in N medium containing dibutyryl cAMP (dbcAMP) and 3-isobutyl-1-methylxanthine (IBMX) did not show protein dephosphorylation, incubation in C medium with dbcAMP/IBMX showed dephosphorylation as well as increased phosphorylation of other proteins (p68, p51, and p29). Finally, calyculin A, a phosphatase inhibitor, prevented dephosphorylation of p96, p90, p64, and p55 but not p105. Based on these data, we propose 2 pathways of protein dephosphorylation that are active during capacitation and independent of cAMP. Together, this provides direct evidence for more complex S/T phosphorylation dynamics than has been previously described for sperm undergoing capacitation.