In this paper the effects of capacitation and fertilisation stimulatingcompounds (heparin, caffeine, glucose, D-penicillamine, bovine serum (BOS),bovine serum albumin (BSA), polyvinyl alcohol (PVA)) were analysed inseveral in vitro fertilisation protocols. Attention was paid to the rateof penetrated oocytes, kinetics of penetration and to polyspermicfertilisation. Cryopreserved bovine sperm and in vitro matured bovineoocytes were used throughout all the fertilisation experiments. As detectedin the first 8 h fertilisation experiment with non-incubated sperm, thesupplementation of medium with heparin, BOS and glucose supported thefertilisation rate most effectively (100%), including the kinetics ofpronuclei formation (52.4%). The absence of BOS resulted in a decreasedfertilisation rate (62.7%) as well as a delay in pronuclei formation(13.6%), similar to that after substitution of heparin with caffeine(73.0% and 25.4%, respectively). The penetration rate in the controlmedium with BOS (without heparin and caffeine) was surprisinglyhigh, especially in medium without glucose (62.2%). The positive effect ofglucose on sperm penetration was observed mainly in a chemically definedmedium with PVA. High polyspermy rates were observed throughout allexperiments in the media containing heparin or caffeine and BOS as themacromolecular component. D-Penicillamine was not shown to be afertilisation-stimulating molecule. However, as detected in the secondexperiment in which oocytes were fertilised with 5 h incubated sperm,its positive effect on the prolongation of a fertile life span ofcryopreserved spermatozoa was significant. The presence of eithercaffeine or heparin in the fertilisation medium (FM) with BOS duringsperm incubation induced tyrosine phosphorylation of an approximately90 kDa protein, detected after 5 h of sperm incubation. The absence ofBOS reduced tyrosine phosphorylation of this protein in fertilisationmedium with heparin. The percentage of motile spermatozoa and those withintact acrosomes were monitored throughout all experiments.