The present study establishes the earliest time point for commitment of cardiac myocyte progenitors at gastrulation and determines the effects of bromodeoxyuridine (BrdU) on postgastrulated committed cardiac progenitor cells at a molecular level. Using immunochemical and reverse transcription/polymerase chain reaction assays for cardiac muscle-specific gene expression, we found that while both pre- and postgastrulated embryonic cells were capable of cardiogenic differentiation at high cell density, only postgastrulated cells exhibited the ability to differentiate at clonal density. These data indicate that while cardiac myocyte differentiation of pregastrulated cells can occur in vitro, cell-cell interactions are necessary for this to happen. Only gastrulated cardiac progenitor cells are able to differentiate in the absence of cell-cell interactions and are therefore both specified and committed. Next, by exposing postgastrulated committed cardiac progenitor cells from embryos at various stages to BrdU, we demonstrated that these cells from the lateral-plate mesoderm vary in their ability to differentiate into cardiac myocytes in vitro. Differentiation of cardiac myocyte progenitor cells from stages 4 and 5 was completely blocked by BrdU, whereas it was not blocked in cells from stages 7 and 8 and cells from stage 6 were varied in their reaction. Analysis of cardiac myogenesis in vivo revealed that cardiac progenitors acquire BrdU resistance as they migrate, postgastrulation, anteriorly along a rostrocaudal axis. The results from these two experiments suggest that while pregastrulated cells exhibit a limited ability to undergo cardiac myocyte differentiation, only postgastrulated anterior lateral-plate mesoderm contains committed cardiac myocyte progenitors and that these committed progenitors are not equivalent in their ability to differentiate.