fruitless (fru) is a pleiotropic gene which produces an array of variant transcription factor isoforms to fulfil a range of developmental and behavioural roles, from the regulation of axonal pathfinding during embryogenesis, all the way to the precise orchestration of individual steps of the Drosophila melanogaster male courtship ritual. Much of the transcriptional differentiation is achieved through the use of multiple promoters, sex-specific splicing and variant C-termini. Alternative splicing at the 3′ end enables generation of transcripts containing one of at least three zinc finger (Zn-F) domains (A, B or C). Due to the close proximity of these Zn-F–encoding exons at the fru locus, almost all extant fru mutants reduce or eliminate expression of all isoforms from a given promoter(s). This has prevented genetic dissection of their individual roles, and limited functional assignment to the zinc finger triumvirate as a whole. Using gene targeting by homologous recombination, this project set out to generate precise null mutations for type-A and -B Fru isoforms in an attempt to determine which aspects of fru function are conferred by these isoforms. Isoform-specifc antibodies were also generated to confirm the loss of individual isoforms within the generated mutants, and to investigate the expression of different isoforms throughout development. Generation of such an antibody to FruB proteins enabled the developmental expression pattern of this isoform to be assessed for the first time, and expression in the male-specific serotonergic neurons of the abdominal ganglion suggested a possible role for male-specifc FruB isoforms in male fertility. Investigation of the developmental expression patterns of FruC revealed a novel immunostaining pattern for this isoform in a group of coalescing cells which appear towards the end of embryonic development. Isolation of specific-isoform mutants was achieved, giving rise to multiple mutant phenotypes, varying in severity between mutant lines. Analysis of mutants lacking type-A and -B Fru isoforms demonstrated the importance male-specific type-A and/or type-B isoforms in establishing male fertility, and suggested essential roles for sex-non-specifc type-A and/or type-B isoforms in the viability and morphology of both sexes.