Using fusions with green fluorescent protein (GFP), we have identified sequences in the polypyrimidine tract binding protein-associated splicing factor (PSF) that are involved in nuclear and subnuclear localization. Like other splicing factors, PSF localizes to the nucleus, is absent from nucleoli, and accumulates in punctate structures within the nucleus referred to as speckles. However, PSF lacks the known speckle localization domains that have been identified in other proteins. Instead, the localization of PSF to speckles is dependent on an RNA recognition motif (RRM). PSF comprises an N-terminal proline- and glutamine-rich domain, two RRMs (RRM1 and RRM2), and a C-terminal region that contains two nuclear localization signals, both of which are required for complete nuclear localization. Deletion of RRM2 led to a complete loss of speckle localization and resulted in diffuse accumulation of PSF in the nucleus, indicating that RRM2 is required for subnuclear localization. Thus, PSF appears to localize to speckles through a novel pathway that is dependent on its second RRM. Consistent with the use of a novel subnuclear targeting pathway, PSF redistributes to perinucleolar clusters upon the addition of a transcription inhibitor whereas other splicing factors display increased localization to speckles in the absence of transcription. A yeast two-hybrid screen identified four-and-a-half LIM-only protein 2 (FHL2) as a potential RRM2 interaction partner, indicating a possible role for zinc-finger or LIM domains in the localization of splicing factors to subnuclear speckles.