The genetic organization of interval 62B3-4 to 62D3-4 on the Drosophila third chromosome was investigated. The region (designated DRE) includes four known loci: Roughened (R; 3-1.4), defined by a dominant mutation disrupting eye morphology; the nonvital locus Aprt, structural gene for adenine phosphoribosyltransferase; Dras3, a homolog of the vertebrate ras oncogene; and 1(3)ecdysoneless (1(3)ecd), a gene that has been implicated in the regulation of larval molting hormone (ecdysteroid) synthesis. Overlapping chromosomal deletions of the region were generated by gamma-ray-induced reversion of the R mutation. Recessive lethal mutations were isolated based upon failure to complement the recessive lethality of Df(3L)RR2, a deletion of the DRE region that removes 16-18 polytene chromosome bands. A total of 117 mutations were isolated following ethyl methanesulfonate and gamma-ray mutagenesis. These and two additional define 13 lethal complementation groups. Mutations at two loci were recovered at disproportionately high rates. One of these loci is preferentially sensitive to radiation-induced mutational alterations. Additionally, an unusually low recovery rate for cytologically detectable rearrangement breakpoints within the gamma-ray-sensitive locus suggests that an interval of the DRE region closely linked to the R locus may be dominantly sensitive to position effects. Lethal phase analysis of mutant hemizygotes indicates that a high proportion of DRE-region loci (11 of 13) are necessary for larval development. Mutations in five loci cause predominantly first-instar larval lethality, while mutations in four other loci cause predominantly second-instar lethality. Mutations in two loci cause late-larval lethality associated with abnormal imaginal disc development. A temperature-sensitive allele of one newly identified complementation group blocks ecdysteroid-induced pupariation. This developmental block is overcome by dietary 20-hydroxyecdysone, suggesting that a second locus in the region in addition to l(3)ecd may play a role in the regulation of late larval ecdysteroid levels.