Polypyrimidine tract-binding protein (PTB) is involved in pre-mRNA splicing and internal ribosomal entry site (IRES)-dependent translation. In order to identify cellular protein(s) interacting with PTB, we performed a yeast two-hybrid screening. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) was identified as a PTB-binding protein. The interaction between PTB and hnRNP L was confirmed in an in vitro binding assay. Both PTB and hnRNP L were found to localize in the nucleoplasm, excepting the nucleoli, in HeLa cells by the green fluorescent protein (GFP)-fused protein detection method. The N-terminal half of PTB (aa 1-329) and most of hnRNP L (aa 141-558) is required for the interaction between PTB and hnRNP L. (C) 1998 Federation of European Biochemical Societies.