The claret (ca) locus in Drosophila encodes products that are needed both for wild-type eyecolor and for correct meiotic chromosome segregation. Mutants described previously provide evidence that two mutationally independent coding regions are present at ca. We have recovered six new P element-induced and one spontaneous ca mutant. Four of these new mutants affect both eyecolor and chromosome segregation. The high frequency of co-mutation of these two functions suggests that the corresponding genes are closely adjacent to one another. We recovered genomic DNA sequences corresponding to the ca locus by chromosome walking, and showed using revertant analysis that the cloned region encodes ca+. Transformation experiments demonstrate that the mutant effect resulting in meiotic chromosome non-disjunction (nd) and loss is fully rescued by DNA from the cloned region. Two RNAs of 7.4 and 2.2 kb have been identified by Northern blot analysis as the putative eyecolor and segregational products. Expression of the RNAs with respect to males and females, and their presence or absence in ca and nd mutants indicate that the 7.4 kb RNA corresponds to the product needed for wild-type eyecolor and the 2.2 kb RNA is the product required for normal chromosome segregation. These RNAs are transcribed in opposite directions to one another. Alleles that affect both eyecolor and chromosome segregation are deletion mutants that affect both transcripts. Thus, the putative eyecolor and segregational products are encoded by separate genes. Mutants that affect both eyecolor and chromosome segregation apparently do so because they delete essential regions of both genes.