Two experiments were performed to assess the effect of supplemental protein source on sheep embryo development in vitro when incubated in synthetic oviduct fluid medium (SOF). In Experiment 1, a 2 × 3 factorial ( N = 215) design examined two serum sources (20% v/v human serum (HS) vs. sheep serum (SS)) and three incubation vessels (multiwells, containing 500 μl media (MW); microwells, containing 50 μl media (μW); microdrops under paraffin oil, containing 50 μl media (μD)). One-cell embryos were incubated for approximately 144 h at 39°C under humidified 5% CO 2, 5% O 2, 90% N 2. Incubation in medium containing HS resulted in more blastocysts (81 vs. 60%, P<0.001), expanded blastocysts (79 vs. 45%, P<0.001) and hatched blastocysts (51 vs. 22%, P<0.001), and more cells were present in blastocysts ( P<0.01). More blastocysts had hatched ( P<0.05) when incubated in MW compared with μW or μD. In Experiment 2, a 2 × 2 × 2 (+1) factorial ( N = 135) design examined two macromolecules (human serum albumin (HSA) or polyvinyl alcohol) and the presence or absence of two medium additives (minimal essential medium (MEM) non-essential amino acids (NEAA) and MEM essential amino acids plus MEM vitamins (EAA&V)). An additional group, comprising SOF supplemented with 20% HS, was included. Polyvinyl alcohol supplemented media supported limited development with only four out of 61 embryos reaching the 16-cell stage. Human serum albumin supplemented medium supported development, especially if further supplemented with NEAA ( P<0.05). There was no difference for the proportion of embryos developing to blastocyst stage between HS supplemented SOF and HSA + NEAA supplemented SOF, but blastocysts in HS had significantly more cells ( P<0.001) and a greater proportion had hatched (41 vs. 0%). The addition of EAA&V inhibited development beyond the 16-cell stage ( P<0.05). The results demonstrate that human serum is a very effective supplement for sheep embryo culture and that factor(s) in the serum, other than albumin, promote development.