This study aimed to determine the most reliable method to evaluate capacitation of buffalo frozen/thawed sperm. Frozen/thawed sperm were incubated in TALP medium in absence of capacitating agents (control) and in presence of 10 μg/ml heparin for 2 and 4 hrs. Capacitation was assessed by Trypan blue/Giemsa after lysophosphatidylecholine (LPC) exposure, chlortetracycline (CTC) fluorescence assay and immune-localization of tyrosine phosphorylated protein. Furthermore, we evaluated the effect of heparin on penetration, cleavage rates and kinetics of embryo development after heterologous IVF. The percentage of LPC-induced acrosome reacted (AR)-sperm increased (P