Abstract Diepoxybutane-induced mutations of the Drosophila zeste locus were generated in an effort to obtain a null allele. Of 33 mutations of this X-linked gene isolated, 16 were associated with multilocus deletions of zeste and adjacent complementation groups, while the remainder were defects restricted to zeste undetectable by Southern blot analysis. Two of these multilocus deletions (Df(1)zdeb3 and Df(1)zdeb92) were employed in the synthesis of females completely deleted for zeste. Such "zesteless" flies were produced, though at frequencies lower than Mendelian expectations. zeste-deleted females are fertile, and can give rise to zeste-deleted female progeny. These results demonstrate that the product of the zeste gene is not essential to viability or to female fertility, even if absent both as a maternal contribution and as a product of the zygotic genome. However, the possibility that zeste may influence relative viability cannot be excluded. In spite of previous in vitro indications that the zeste protein may activate transcription of the Ultrabithorax (Ubx) gene, zeste -deleted flies are Ubx+ in phenotype. This suggests that the zeste protein normally is either a very weak transcription factor, or that its function can be substituted by that of other regulatory proteins.