We analyzed the roles of the three domains of a Drosophila hnRNP A1 homolog by expression of wild-type and mutant versions of HRB87F/hrp36 in Drosophila melanogaster. HRB87F/hrp36 is one of two Drosophila proteins that is most similar to mammalian A1 hnRNP, and like A1, consists of two copies of the RNA-binding domain (RBD) motif followed by a glycine-rich domain (GRD). The role of the domains in nuclear localization and RNA binding to polytene chromosomal sites was determined. RBD-1 and the GRD were largely responsible for both the cellular location of the protein and for the typical chromosomal distribution pattern of the protein at sites of Pol II transcription. RBD-1 also provided a role in the exon-skipping activity of the protein that was not provided by RBD-2. On the other hand, RBD-2 and the GRD were responsible for the very limited chromosomal distribution pattern seen upon heat shock, when HRB87F/hrp36 is sequestered at heat-shock puff 93D, which encodes a long nucleus-restricted RNA. Thus, these studies indicate that the two RBDs function independently of each other but in concert with the GRD. In addition, the self-association property of the GRD was strikingly evident in these overexpressed proteins.