The pelle gene is required for the nuclear import of dorsal protein that establishes dorsoventral polarity in Drosophila embryos. We report here the genetic mapping and molecular characterization of pelle. DNA sequence analysis revealed that pelle encodes a protein of 501 amino acids, the last 292 of which comprise a protein kinase catalytic domain. Microinjection of in vitro synthesized transcripts containing site-directed mutations indicates that the kinase catalytic domain is required for biological activity. This domain is most similar to that of the rat and mos protein kinases and is predicted to have a serine and threonine specificity. These results provide direct evidence for the role of phosphorylation in the in vivo regulation of a rel-like transcription factor.