The complete amino acid sequence of Xenopus laevis nonmuscle myosin heavy chain B (MHC-B) has been deduced from overlapping cDNA clones isolated from an XTC cell library. RNA blots of various developmental stages, adult tissues, and XTC cells detect a single transcript of 7.5 kb which is expressed at similar levels throughout development. MHC-B mRNA was detected in XTC cells, heart, lung, spleen, and brain, at lower levels in ovary, testis, pancreas, stomach, liver, and eye, but not in kidney and skeletal muscle. Protein expression in adult tissues, as detected by immunoblot analysis, correlates well with mRNA expression. In chickens and humans, a fraction of the mRNA encoding the MHC-B isoform was found previously to contain a 10-amino acid insert at amino acid 211 near the ATP-binding site. As reported elsewhere, in the chicken this insert-bearing isoform is nervous system-specific. The Xenopus sequence shows a 16-amino acid insertion at the same position; 7 of 16 residues are identical to those in the chicken and human insertion, and these identical residues include a consensus target sequence for cyclin-p34cdc2 kinase. In contrast to chicken, all frog tissues and embryonic stages tested contained the insert-bearing form, and no evidence for a non-insert-bearing MHC-B isoform was found in Xenopus.