DNA-mediated gene transfection using an alpha-casein minigene cloned into a bovine papilloma virus (BPV)-based neomycin-selectable expression vector has been employed to study the mechanisms by which hormonal and cell-substratum interactions regulate milk protein gene expression. Permanently transformed clones and pooled populations of normal midpregnant mouse mammary epithelial cells (COMMA-D) containing the minigene express an authentic rat alpha-casein mRNA, as well as a series of larger cytoplasmic RNA transcripts. These transcripts are correctly initiated and spliced; however, a large proportion also contain additional sequences at the 3'-end. Constitutive expression of the minigene in the absence of PRL and glucocorticoids in COMMA-D cells grown on floating type I collagen gels is observed. Thus, the minigene-BPV construct apparently overrides the normal posttranscriptional regulatory mechanisms which are responsible for the expression of unstable casein gene transcripts in the absence of PRL and glucocorticoids. In contrast, this minigene-BPV construct is regulated appropriately by cell-substratum interactions in pooled transfectants. Minigene expression is undetectable when pooled transfectants are plated on a plastic substratum, and readily detectable when cells are grown on floating type I collagen gels. Thus, hormones and cell-substratum interactions may regulate different steps in the same differentiation pathway leading to increased casein gene expression.