Mutations at the Drosophila melanogaster locus female sterile (1) homeotic (fs(1)h) result in segmental abnormalities including missing organs and homeotic transformations in the progeny of mutant mothers. Homeotic transformations are enhanced when the zygotes carry one of several third chromosome mutations, specifically alleles or deficiencies of the trithorax (trx) locus, also called Regulator-of-bithorax, and some alleles of bithorax complex (BX-C) genes. These observations suggest that maternally derived fs(1)h+ product is required, in interaction with trx and BX-C genes, for normal segment specification. The fs(1)h gene and an adjacent gene, lethal (1) myospheroid (l(1)mys), have been cloned by chromosomal walking. Mutations of fs(1)h were found within a 13-kb stretch of DNA. Poly(A)+ RNAs migrating as a doublet at 7.6 kb and a single band at 5.9 kb, which are homologous to the fs(1)h+ chromosomal region, are found in ovaries and early embryos. The largest RNAs are derived from a 20-kb chromosomal region encompassing the sites of all mapped fs(1)h alleles.