Ovulated rabbit oocytes were fertilized in vitro in chemically defined media supplemented with bovine serum albumin and either cultured up to the expanding blastocyst stage or transferred to recipients after varying periods of culture. Embryos transferred after up to 72 hours of in vitro culture were born as viable young. Oocytes from young virgin does were superior to oocytes from nonvirgin does for the purpose of in vitro fertilization (54% versus 26% fertilized, P less than 0.01). Capacitated sperm from artificially inseminated capacitators resulted in fertilization rates slightly lower than those from naturally mated does (46% versus 57% fertilized, P less than 0.025). Removal of cumulus and corona cells from oocytes with hyaluronidase and repeated aspiration through a fine pipette resulted in lowered fertilization rates (51% versus 73%, P less than 0.025). Linbro Disposo Tray wells were as good as glass tissue-culture dishes for the in vitro mixing of gametes and were more convenient to use. Modified Ham's F10 medium was used to culture the in vitro-fertilized embryos. However, when a modified Brackett's medium was used instead of modified Ham's F10 for the initial 4-hour period after mixing gametes, more oocytes were fertilized (52% versus 28%, P less than 0.01).