We investigated the expression of myosin heavy chain (MHC) isoenzymes in embryonic rat ventricles cultured in the anterior eye chamber of an adult rat. In oculo, these grafts beat and mature in an environment where the hormonal milieu can be manipulated. S1 nuclease protection assays were performed on pooled samples of ventricle grafts and compared to normally growing ventricles. At the time of grafting (embryonic day 12, E-12), 23 ± 4% of the MHC mRNA was of theα isoform. While the proportion of ventricular α-MHC mRNA did not increase in utero, embryonic ventricles cultured in oculo showed a rapid increase in the relative amount of α-MHC mRNA expression (to 84 ± 10% by 3 days and 86 ± 5% by 8 days in oculo). α-MHC mRNA expression predominated through 8 weeks of culture in oculo, being 76% at 8 weeks in oculo. Additional experiments were performed to determine whether the rapid conversion to α-MHC expression resulted from exposure to adult levels of testosterone or thyroid hormone. Reduction of testosterone exposure to non-detectable levels by host orchiectomy did not affect the rapid conversion to α-MHC mRNA expression. Exposure to a hypothyroid milieu (i.e., PTU-treated hosts) decreased but did not prevent the conversion from β- to α-MHC mRNA expression at 8 days in oculo; with 83% of the MHC mRNA being of the α isoform in hypothyroid hosts compared to 95% in euthyroid hosts. After 8 weeks of culture in hypothyroid hosts, however, α-MHC mRNA expression was undetectable in grafted ventricles. These data suggest that E-12 myocardial grafts respond to the hormonal milieu of an adult rat with rapid conversion from β- to α-MHC mRNA expression and that α-MHC expression in early developing heart may show reduced sensitivity to downward modulation by a hypothyroid hormonal milieu. © 1995 Academic Press Limited.