Expanded blastocysts collected from superovulated Sarda ewes were divided at random into four groups for culture in a simple medium that does not support blastocyst hatching (CZB) or a complex medium that is permissive to hatching (TCM 199), with or without vasoactive intestinal peptide (VIP), a known embryo mitogenic peptide. Plasminogen activator (PA) secretion after 24 h of culture, and the number of cells, diameter of blastocysts and hatching rate after 48 h of culture were compared. The results showed an increase in hatching rate (78.6 v. 6.7%; P<0.01), diameter and number of cells (220.89 v. 210.44 microm, P<0.01 and 246 v. 232, P<0.01 respectively) and caseinolytic areas (1.33 v. 0.92 cm, P<0.01) of blastocysts cultured in TCM 199 compared with those cultured in CZB. Supplementation of the culture media with VIP increased these parameters in CZB (P<0.01) and partially in TCM 199. In particular, cell number, diameter and PA activity were significantly higher (P<0.01) after culture with VIP in both media. Immunoneutralization of exogenous VIP in culture with anti-VIP antibody caused a decrease in the hatching rate (P<0.01) of embryos cultured in medium with VIP, similar to the rate in unsupplemented CZB (P<0.01). These results suggest a receptor-mediated response. In immunohistochemical studies, VIP was shown to bind receptors in hatched blastocysts demonstrating the VIP-receptor interaction, and VIP receptors of approximately 150 kDa were revealed by electrophoretic studies. In conclusion, ovine preimplantation embryos exhibit VIP receptors, providing a basis for a receptor-mediated influence of VIP in the hatching of ovine blastocysts.