The 58-kDa inhibitor of the interferon-induced double-stranded RNA-activated protein kinase (PKR) is a cellular protein that is activated during influenza virus infection to down-regulate the activity of PKR. This study was initiated to further our understanding of the inhibitor which, when overproduced, has the capacity to malignantly transform cells. We report here the isolation and characterization of cDNA clones encoding the inhibitor, designated p58, from human HeLa and mouse NIH 3T3 cells. The human and mouse p58 cDNAs were 6.5 and 1.6 kb in length, respectively. Surprisingly, the deduced amino acid sequences of the human and mouse p58 were 96% identical, indicating a remarkably high degree of conservation between species. An examination of p58 mRNA expression in human tissues revealed a 6.5-kb transcript in all tissues examined, with a particularly high level of expression present in the pancreas and liver, and also in certain leukemic cell lines. Similarly, p58 expression was detected in all mouse tissues examined, with the highest level of expression found in liver. In contrast to human tissues, three p58 transcripts of approximately 1.7, 3.3 and 5.4 kb were observed in mouse tissues, suggesting that p58 expression may be regulated differently in human and mouse cells. Western blot analysis of subcellular fractions and indirect immunofluorescence analysis of intact cells revealed that p58 was found predominantly in the cytoplasm, consistent with its function as an inhibitor of PKR, which is also a predominantly cytoplasmic protein.