Embryoid bodies (EBs) are obtained when mouse pluripotential embryonic stem cells are grown in the absence of an embryonic fibroblast feeder layer. Seven- to 9-day-old EBs undergo rhythmical, spontaneous contractions and express the appropriate tissue- and developmental stage-specific cardiac and skeletal myosin heavy chain (MHC) genes. To study the expression patterns of these MHC genes in vitro we isolated and partially sequenced the cDNAs expressed in EBs such that specific oligonucleotides suitable for polymerase chain reaction analyses and appropriate riboprobes for in situ hybridizations could be made. The data show the beta-cardiac gene is expressed first during EB development (days 3 and 4), and alpha-cardiac gene expression begins at approximately day 8. A similar pattern of expression is also detected during mouse embryogenesis in utero. Only those EBs that expressed both the alpha- and beta-cardiac transcripts contracted. In situ hybridization of EBs using riboprobes shows that the spatial distribution of the cardiac MHC transcripts differs. No expression of the genes was detected in day 8 or older nonbeating EBs. These data suggest that developing EBs closely mimic the pre- and early postsomitic patterns of in vivo expression of the cardiac MHC genes and thus provide a useful system in which to study early aspects of mammalian cardiogenesis.