The ability of pure viral and cellular single-stranded (ss) RNAs to activate the interferon-induced, double-stranded (ds) RNA-dependent P1 /eIF-2 protein kinase purified from human amnion U cells was examined. In addition to the well-established activation of P1 kinase autophosphorylation in vitro by reovirus genome dsRNA, the P1 kinase was also efficiently activated by certain reovirus ssRNAs. The reovirus s1 mRNA was a potent activator of the kinase. By contrast, the reovirus s4 mRNA was a poor activator of the kinase. Likewise, adenovirus VAI RNA, transfer RNA, 5 S ribosomal RNA, and rabbit globin mRNA were not activators or were very poor activators of the purified Pl/eIF-2 protein kinase. Analysis of hybrid ssRNAs produced between the reovirus s1 and s4 mRNAs revealed that both the 5′ and the 3′ portions of the sl mRNA possessed nucleotide sequences capable of mediating kinase activation. Subsequent deletion analysis of the 5' portion of the s1 mRNA identified a 161-nucleotide region located between positions 416 and 576 which was sufficient for P1 kinase activation. Treatment of reovirus s1 mRNA transcripts with either ssRNA- or dsRNA-specific ribonucleases, but not with heat, destroyed the ability of s1 mRNA transcripts to activate the kinase. These results suggest that P1 kinase autophosphorylation in vitro may be selectively activated by individual ssRNAs in a differential manner, and that a secondary or higher-ordered ssRNA structure(s) may be important in mediating the activation