Previously we have shown that a bovine mammary epithelial cell clone isolated from lactating mammary tissue is able to differentiate morphologically and functionally on a reconstituted basement membrane. Here we show that when cultured on collagen I coated culture inserts, the presence of lactogenic hormones results in an increase in polarized secretion of α-lactalbumin but not α-casein. We conclude that collagen I is sufficient for milk protein gene expression and that culture insert methodology offers a practical alternative to three-dimensional culture systems when α-lactalbumin is used as an indicator of mammary specific function.