Human recombinant ribosomal protein S26 (rpS26) was shown to interact with its pre-mRNA intron I and mRNA fragment. Endogenous rpS26 in HeLa nuclear extract was also found to bind to the intron I, and with a lower extent to the mRNA fragment. The addition of recombinant rpS26 to the nuclear extract increased the binding largely. The in vitro splicing of an RNA that contained exon I, intron I and part of exon II of the rpS26 pre-mRNA yielded conventional and alternative mRNAs. Recombinant rpS26 was found to suppress the formation of both mRNAs. Sites of the pre-mRNA involved in the binding to rpS26 were detected by toe-printing. Nucleotides that caused a stop (pause) of the reverse transcription formed two clusters on the RNA secondary structure. One cluster including A69, A287 and A303 arranged the conventional 3' site of splicing, another one including A131, A136, G156, A166 and A264 arranged the alternative 3' site of splicing.