The effects of alpha-amanitin on cumulus enclosed and denuded porcine oocytes exposed to this heterogeneous nuclear RNA (hnRNA) synthesis inhibitor at different times (0, 0.5, 1, 2, 4, 6, 8, 12 and 20 h) after the start of culture was investigated. A further objective was to determine the sequence of nuclear progression after removing the inhibitor. The addition of 10 micrograms alpha-amanitin ml-1 to a gonadotrophin containing medium (10 iu PMSG ml-1 in TCM 199) at 0, 0.5 or 1 h after the onset of culture prevented cumulus expansion, and only 4.4, 6.4 and 3.5% of oocytes underwent germinal vesicle breakdown. This inhibitory effect was considerably reduced by delaying the addition of the drug to the culture medium for 2-8 h (2 h: 34.9%, 4 h: 53.5%, 6 h: 46.9%, 8 h: 59.2% germinal vesicle breakdown), and no inhibition of nuclear maturation was observed when alpha-amanitin was added after 12 or 20 h following explantation of the oocytes. When cumulus-oocyte complexes were cultured for > or = 2 h in inhibitor-free medium and then transferred to medium supplemented with alpha-amanitin, full cumulus expansion was observed in all cases, at the end of the 44 h culture. Denudation of the oocytes before culture in either medium supplemented with alpha-amanitin or microinjection of alpha-amanitin into the ooplasm at concentrations of 1.0 and 10.0 mg ml-1 remained without any effect on nuclear progression.(ABSTRACT TRUNCATED AT 250 WORDS)