A gal4-containing enhancer-trap called C309 was previously shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shi(TS)). We extended these manipulations to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shi(TS) at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, we found a C309/tra(F) combination also to induce high levels of courtship between pairs of males and "chaining" behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRU(M) protein is regulated by TRA, we hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fru(MIR)) would mimic the effects of tra(F); but C309/fru(MIR) males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRU(M) in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of tra(F) or fru(MIR) effects on courtship song, because we found C309 to be coexpressed with FRU(M) within the same 10 neuronal clusters in pupae. Thus, we hypothesize that elimination of singing behavior by the C309/shi(TS) combination involves neurons acting downstream of FRU(M) cells.