Genomic and P1 clone DNAs of Drosophila virilis were analyzed to determine the structure and organization of histone genes in this species. The species contains unique and variable repeat types, in comparison with the related species Drosophila melanogaster, with quartet repeats lacking the H1 gene and multi-length variant quintet repeats containing the H1 gene. Unexpectedly, the H1-containing repeats are highly polymorphic in length, and thus not in a strict tandem arrangement, while the H1-less repeats are very uniform and tandemly reiterated. Despite such differences, the relative positions and transcriptional polarities of the histone gene subtypes of one subcloned quintet are similar to the major histone repeat type of D. melanogaster. For the first time, the histone H1 gene has been shown to be associated with other histone gene subtypes and is present at both chromosomal loci. DNA sequence variants of the H1 gene have been mapped to individual P1 clones and found to be in a partitioned organization. The P1 cloning system has proved useful in completely retrieving a complex repetitive locus in vitro and in examining the structure and organization of the histone genes of D. virilis.