We have isolated chromosomal segments of D. melanogaster DNA that encode the alpha, beta and gamma polypeptides of the major larval serum protein (LSP1) from libraries of recombinant phage. The recombinant DNAs hybridize in situ to polytene chromosome regions 11A, 21D of 61A, consistent with the known cytogenetic positions of the alpha, beta and gamma genes, respectively. The three genes each encode a 2.85 kb polyadenylated RNA molecule. We have positioned these coding sequences, by both Southern hybridization analysis and R loop mapping. Heteroduplex analysis reveals comparatively close homology between the alpha and beta genes, less homology between the beta and gamma genes and least homology between the alpha and gamma genes.