Immature eggs were recovered from freshly excised ovaries from domestic cats, and initially 931 eggs with a compact cumulus and uniform cytoplasm were cultured in 1 of 15 treatments. Eagle's minimum essential medium containing glutamine and pyruvate was supplemented with 5% (v/v) fetal calf serum (FCS), 4 mg bovine serum albumin (BSA) ml−1 or 2 mg polyvinyl alcohol ml−1 (PVA; non-protein control). Within each of these supplement groups, eggs were cultured with: no hormone; LH + FSH; LH + FSH + oestradiol; or LH + FSH + oestradiol + progesterone. After incubation for 52 h, eggs were inseminated with conspecific fresh spermatozoa, cultured and examined for stage of meiotic maturation and in vitro fertilization (IVF). There were fewer (P <0.05) eggs maturing to metaphase II in vitro in FCS compared with BSA or PVA, the last two treatments producing similar (P < 0.05) results. Gonadotrophins in concert with oestradiol or oestradiol + progesterone improved the incidence of maturation (P ≤ 0.01) compared with no added hormones. The incidence of fertilization and cleavage in vitro ranged from 5.2 to 33.9% and varied (P < 0.05) with hormone subtreatment. Adding FSH + LH + oestradiol consistently increased the incidence of IVF approximately twofold compared with controls with no added hormones. Although it inhibited the ability of eggs to reach metaphase II, FCS in the presence of gonadotrophins and oestradiol allowed > 60% of mature eggs to fertilize in vitro (P < 0.05, compared with PVA and BSA). Inhibitory effects on egg maturation were further evaluated by testing four FCS batches from three commercial sources against BSA. All FCS batches were clearly inhibitory, as 60.7% of treated eggs arrested at the germinal vesicle stage (21.1% of BSA-treated eggs, P < 0.001). Dialysis of FCS eliminated a significant proportion of the inhibition; 69 of 158 eggs (43.7%) matured compared with 22 of 135 (16.3%) in complete or 23 of 160 (14.4%) in recombined serum. In summary, FCS appears to exert a paradoxical effect in this system, inhibiting maturation in vitro (apparently due to small molecular weight components), but promoting IVF of mature eggs if gonadotrophins and oestradiol are present. Although hormone supplementation enhanced the ability of in vitro matured eggs to fertilize and cleave, neither protein source nor the hormone treatments tested here appear responsible for the consistently low incidence of fertilization in cat eggs matured in vitro.