We determined the actin isotypes encoded by 30 actin cDNA clones previously isolated from an adult human muscle cDNA library. Using 3' untranslated region probes derived from alpha-skeletal, beta- and gamma-actin cDNAs and from an alpha-cardiac actin genomic clone, we showed that 28 of the cDNAs correspond to alpha-skeletal actin transcripts. Unexpectedly, however, the remaining two cDNA clones proved to derive from alpha-cardiac actin mRNA. Sequence analysis confirmed that the two skeletal muscle alpha-cardiac actin cDNAs are derived from transcripts of the cloned alpha-cardiac actin gene. Direct measurements of actin isotype mRNA expression in human skeletal muscle showed that alpha-cardiac actin mRNA is expressed at 5% the level of alpha-skeletal actin. Furthermore, the alpha-cardiac actin gene expressed in skeletal muscle is the same gene which produces alpha-cardiac actin mRNA in the human heart. Of equal surprise, we found that alpha-skeletal actin mRNA accounts for about half of the total actin mRNA in adult heart. Comparison of total actin mRNA levels in adult skeletal muscle and adult heart revealed that the steady-state levels in skeletal muscle are about twofold greater, per microgram of total cellular RNA, than those in heart. Thus, in skeletal muscle and in heart, both of the sarcomeric actin mRNA isotypes are quite abundant transcripts. We conclude that alpha-skeletal and alpha-cardiac actin genes are coexpressed as an actin pair in human adult striated muscles. Since the smooth-muscle actins (aortic and stomach) and the cytoplasmic actins (beta and gamma) are known to be coexpressed in smooth muscle and nonmuscle cells, respectively, we postulate that coexpression of actin pairs may be a common feature of mammalian actin gene expression in all tissues.