Effects of extracellular matrix proteins and tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) on bovine inner cell mass (ICM) outgrowth and proteinase production in vitro were determined. Inner cell masses were isolated immunosurgically from day 7 embryos (day 0 = onset of estrus) and cultured for 96 h. In experiment 1, cellular outgrowth and gelatinase production were evaluated for ICM cultured on collagen IV, fibronectin, or laminin. More (P < 0.05) ICM generated cellular outgrowth on fibronectin (71%), compared with collagen IV (0%) or laminin (15%). Inner cell mass and outgrowth areas were greatest (P < 0.05) on fibronectin after 96 h of culture, compared with laminin. Although the incidence of cellular outgrowth on laminin was limited, numbers of cells in outgrowths supported by laminin were similar (P > 0.10) to fibronectin except at 72 h of culture, where more (P < 0.05) cells were in laminin than in fibronectin outgrowths. Gelatinase activity was not detected in conditioned medium. In experiment 2, cellular outgrowth and plasminogen activator production by ICM cultured on fibronectin in medium containing 0 or 10 μg/ml TIMP-2 were evaluated. Inner cell mass and outgrowth areas, and numbers of cells in outgrowths were greater (P < 0.05) in 10 compared with 0 μg/ml TIMP-2 at 96 h of culture. Mean plasminogen activator activity in conditioned medium from ICM cultured in 10 μg/ml TIMP-2 was greater (P < 0.05) compared with 0 μg/ml TIMP-2 (16.2 ± 4.8 versus 6.7 ± 1.4 × 10⁻³ IU/ml, respectively). These results demonstrate that cellular outgrowth from bovine ICM is supported by fibronectin and is stimulated by TIMP-2.