The Drosophila ovo gene, which encodes a putative transcription factor (Ovo) with TFIIIA-like zinc fingers, is required for female germline survival and proper oogenesis. Three dominant female-sterile ovoD mutations cause ovarian abnormalities that define an allelic series, with ovoD1 displaying the stronger phenotype and ovoD3 the weaker. We report here that all three ovoD mutations are point mutations that create new in-frame methionine codons in the 5' part of ovo. There are two types of overlapping ovo transcription units, ovo alpha and ovo beta. By using various ovo-lacZ reporter genes, we determined that the long Ovo isoforms starting at methionine M1, present in transcripts ovo alpha, are expressed at low levels only in mature oocytes. Short Ovo isoforms are translated from methionine M373, the first in-frame start codon present in transcript ovo beta, and correspond to the activity defined by recessive loss of function ovo mutations. The new AUGs created in ovoD mutations all are located upstream of the M373 initiation site. Our results support the hypothesis that they can substitute for M373 as translation starts and initiate the synthesis of Ovo proteins that have extra amino acids at their N termini. We propose that premature expression of long Ovo protein isoforms occurs in ovoD mutants and interferes with wild-type Ovo function in controlling female germline differentiation.