The ability of undifferentiated cardiogenic mesoderm to generate diversified myogenic phenotypes was assayed in a minimal culture system. During cardiogenesis in vivo, the anterior and posterior segments of the avian heart have distinct patterns of contractile protein gene expression when they first differentiate. To assess the potential of undifferentiated cardiogenic tissue to diversify into distinct anterior and posterior lineages prior to heart formation, cardiogenic mesoderm and endoderm were removed together from the embryo at Hamburger and Hamilton stages 4-8. Explants from each of these stages differentiated in defined medium as indicated by the expression of muscle-specific genes. However, the ability to express the atrial-specific myosin heavy chain (AMHC) mRNA was confined to posterior cardiac progenitors. Diversification was not dependent on anterior endoderm, suggesting that inductive interactions between the mesoderm and endoderm are not necessary to maintain diversified cardiac lineages after stage 4. The diversified potential of explanted cardiogenic tissue was altered with retinoic acid treatment, resulting in the activation of AMHC1 gene expression in the anterior progenitors. Anterior cardiogenic cells removed from the embryo at stage 8, when the heart begins to differentiate in vivo, are not susceptible to the alteration of diversified phenotype by retinoic acid treatment. Therefore, the potential to form distinct cardiomyogenic cell lineages is present in the anterior lateral plate mesoderm soon after gastrulation and the maturation of these lineages in a positionally dependent manner is maintained in a simple defined culture system in vitro.