Oocyte-cumulus cell complex maturation and fertilization potential of 4700 bovine oocytes in the presence of various intrafollicular regulators were studied. Endpoints for oocyte maturation included cumulus cell expansion, retention of [3H]hyaluronic acid, elevated cyclic AMP concentrations, and germinal vesicle breakdown. Media supplemented with FSH and fetal bovine serum stimulated oocyte maturation and fertilization. Heparin and mixed glycosaminoglycans (40% heparan sulfate and 60% dermatan sulfate) stimulated cumulus cell expansion and increased [3H]hyaluronic acid but decreased cyclic AMP and reduced percentages of oocytes with germinal vesicle breakdown at 12 h. Oocytes cultured in heparin or mixed glycosaminoglycans were incapable of fertilization. Addition of serum to oocytes treated with heparin decreased cumulus cell expansion. Addition of FSH to heparin and serum cultures did not overcome the inhibitory effect of serum on heparin-induced expansion. Dermatan sulfate failed to stimulate expansion of cumuli but resulted in cells retaining [3H]hyaluronic acid and elevated cyclic AMP content of oocytes. Dermatan sulfate did not affect the percentage of germinal vesicle breakdown at 12 h. Nitrogen-desulfated heparin did not promote any signs of oocyte maturation. These results demonstrate that heparin and mixed glycosaminoglycans stimulate events related to oocyte maturation but were not capable of preparing oocytes for fertilization. The addition of serum or desulfation of heparin inhibited heparin effects on oocyte maturation.