We have constructed a double-stranded cDNA library using total poly(A)-containing RNA extracted from 8-day lactating rat mammary gland and have utilized this library to isolate clones for each of the four major milk proteins. These four cDNA clones, representing the three major rat caseins and alpha-lactalbumin, were initially identified by colony hybridization with labeled cDNA probes synthesized from individual mRNA fractions purified by preparative gel electrophoresis. Additional characterization was accomplished by hybridizing individual clones labeled with 32P by nick translation to a Northern gel blot of an enriched fraction of the four major milk protein mRNAs. The individual mRNAs were clearly resolved by electrophoresis on fully denaturing methylmercury hydroxide agarose gels. The identity of each milk protein clone was further established by the location of unique restriction enzyme sites within each clone. Final identification of each clone was performed by hybrid-arrested cell-free translation. The sizes of the milk protein cDNA clones ranged frm 70% for the alpha-lactalbumin gene to essentially full length for the gamma-casein gene, in comparison to their respective mRNAs. This represents the first isolation of a family of peptide hormone-responsive genes.