We have investigated the factor(s) present in serum that stimulate cumulus expansion (accumulation of hyaluronic acid in the intercellular space of cumulus cells) and male pronucleus (mPN) formation in porcine oocytes matured and fertilized in vitro. After 24 hr of culture, oocyte-cumulus complexes that matured in the serum of pig, cow, rabbit and rat showed an expansion of the surrounding cumulus cell matrices. The cumulus expansion stimulating effect is not species-specific. Porcine serum was fractionated by ultracentrifugation at 220,000 x g at 10 degrees C for 48 hr, resulting in four fractions. The oocyte-cumulus complexes that matured in the 2nd fraction exhibited a marked expansion of the cumulus cell layers, including the corona radiata. Complexes cultured in the remaining fractions, however, showed very little or no expansion. There was no expansion of the oocytectomized complexes matured in the 2nd fraction. Serum was not effective in the formation of the male pronucleus. The active factor(s) was heat stable at 100 degrees C for 30 min and showed a relatively strong activity at a molecular weight of > 1.0 and < 6.5 kDa. These results indicate that serum contains a low molecular weight, heat stable factor(s) that acts on the oocyte to stimulate cumulus expansion.