The rotund (rn) gene in Drosophila melanogaster codes for a RacGTPase-activating protein, RnRacGAP. Cellular studies have shown that RacGAP proteins function as negative regulators of substrate Rac proteins which, in turn, control the localization and polymerization state of actin within the cell. Previous sequence analysis of rn genomic DNA and incomplete cDNA clones suggested that at least two differentially spliced forms of the transcript exist, rnRacGAP(1) and rnRacGAP(2). Using nested reverse transcription-polymerase chain reaction (RT-PCR) methods, we have cloned missing exon and intron sequences, and detected differences between rnRacGAP(1) and rnRacGAP(2) involving 24 nucleotides (nt) of coding sequences and 119 nt of 3'UTR. This translates to a difference of seven amino acids at the C-termini of the polypeptide products. Utilization, in RT-PCR analysis, of form-specific primers provided a simple assay for the tissue specificity of expression of the two forms. rnRacGAP(1) is the predominant species in the testes and is expressed at a low level in the ovary and somatic tissues. rnRacGAP(2) is only very weakly expressed and is detectable solely in the testes.