Heterogeneous nuclear RNA-binding proteins, hnRNPs, have been implicated in nuclear export of mRNAs in organisms from yeast to humans. A germ-line mutation in a Drosophila hnRNP, Squid (Sqd)/hrp40, causes female sterility as a result of mislocalization of gurken (grk) mRNA during oogenesis. Alternative splicing produces three isoforms, SqdA, SqdB, and SqdS. Here we show that these isoforms are not equivalent; SqdA and SqdS perform overlapping but nonidentical functions in grk mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. We show that this differential nuclear accumulation is correlated with a differential association with the nuclear import protein Transportin. Finally, we provide evidence that grk mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. These results demonstrate the isoform-specific contributions of individual hnRNP proteins in the regulation of a specific mRNA. Moreover, these data suggest a novel role for hnRNPs in localization and translational regulation of mRNAs.