The fruitless (fru) gene acts sex-nonspecifically in the development of the embryonic central nervous system (CNS) and has sex as well as sex-nonspecific functions in the development of the adult CNS. In the embryo, sex-nonspecific fru mRNAs and proteins are widely expressed during neurogenesis and present in both neurons and glia. To assess whether the fru gene played any role in fate determination of neuronal precursors and neurons, we examined the development of Eve-positive (Eve(+)) GMCs and neurons in fru mutants. In fru mutant embryos in which most or all fru transcripts were eliminated, the normal complement of Eve(+) neurons was present initially, but some neurons were unable to maintain their Eve-expression. Concomitantly, a subset of Eve(+) neurons also showed inappropriate expression of the glial marker, reversed polarity. In addition, neurons that normally do not express Eve became Eve(+) in these fru mutants. These defects were rescued in fru mutant embryos expressing specific fru transgenes under the control of the sca-GAL4 and elav-GAL4 drivers. These phenotypic analyses and rescue experiments provide evidence that one of the sex-nonspecific functions of the fru gene is the maintenance of neuronal identity rather than establishment of a neuron's initial fate.