PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma that is able to preserve, in vitro, the viability, motility and mitochondrial activity of highly-extended boar spermatozoa. However, a relationship between the protective effects of the heterodimer and sperm capacitation is still unclear. The present study investigated the effect of the PSP-I/PSP-II (1.5 mg/mL) on membrane stability, intracellular calcium concentration ([Ca(2+)](I)) and plasma membrane and acrosome integrity of highly extended boar spermatozoa. Boar spermatozoa were diluted to 1 x 10(6) spermatozoa/mL and incubated at 38 degrees C in Phosphate-buffered saline (PBS) for 10, 30, 60, 120 and 300 min or in modified Tris-buffered medium (mTBM) for 10, 20, 30, 60 and 120 min. After each incubation time, the membrane stability (using Merocyanine-540/Yo-Pro-1), elevation of [Ca(2+)](I) (using Fluo-3-AM/PI) and the sperm plasma membrane and acrosome integrity (using SYBR-14/PI/PE-PNA) were evaluated by flow cytometry. As expected, exposure of the spermatozoa to the PSP-I/PSP-II preserved the plasma membrane and acrosome integrity compared to non-exposed spermatozoa in both media PBS and mTBM (p < .01). The evaluation of membrane stability showed no differences in the percentages of viable sperm with instable plasma membrane in the presence of the PSP-I/PSP-II compared to controls irrespective of the dilution media. The evaluation of the [Ca(2+)](I) levels showed that while spermatozoa incubated in mTBM and exposed to PSP-I/PSP-II had lower [Ca(2+)](I) than controls (39.08% vs. 47.97%, respectively; p < .05), no differences were observed in those samples incubated in PBS. However, a temporal evaluation of the samples showed that a similar proportion of live spermatozoa were able to achieve high levels of [Ca(2+)](I) and membrane instability independent of the presence of PSP-I/PSP-II. In conclusion, PSP-I/PSP-II exert a non-permanent decapacitation effect on highly extended boar spermatozoa that is related with a delay in the increase of [Ca(2+)](I) levels.