The effects of fractionated bovine uterine flushings (BUF) on in vitro mouse embryo development were evaluated. Uterine flushings were nonsurgically collected from six cows and fractionated by ultrafiltration using 1,000 D and 30,000 D molecular weight exclusion limit membranes. Retentates were designated as 1,000 D (1 KR) and 30,000 D retentates (30 KR) and were evaluated for total protein, plasmin, plasmin inhibitor and support of mouse embryo development. Medium with bovine serum albumin (BSA) as a control was fractionated in a similar manner and frozen and thawed to assess any developmental limitations induced by the procedure. Consistent significant relationships, as determined by correlation-regression analysis, between the extent of embryo development and levels of protein, plasmin and plasmin inhibitor were not observed in either 1 KR BUF or 30 KR BUF. The numbers of embryos developing into blastocysts in 1 KR BUF on Days 9, 12, 15, 18 and 21 of the collection period were reduced when compared with the 30 KR BUF (P<0.05). Fewer blastocysts hatched in 1 KR BUF than in 30 KR BUF on Days 12, 15, 18 and 21 of the collection period (P<0.05). Embryo development in medium with 1 KR BSA tended to be superior to development in unfractionated media and in 30 KR BSA at comparable protein levels. No detrimental effects of freezing and thawing culture media on embryo development were observed (P>0.10). These data suggest that luteal phase BUF contain an inhibitor of embryo development, and commercial BSA preparations may possess a small molecular weight contaminant which reduces in vitro embryo survivability.