This laboratory recently reported the occurrence of concentrated deposits of fibroblast growth factor-2 (FGF-2; basic FGF)-like proteins in the sarcoplasm of embryonic cardiac myocytes from the earliest stage of avian heart development (M. H. Parlow, D. L. Bolender, N. P. Kokan-Moore, and J. Lough, Dev. Biol. 146, 139, 1991). To determine the role, if any, of FGF-2 during embryonic cardiogenesis, the proliferative and functional effects of treating cultured anterior lateral plate mesoderm from Hamburger-Hamilton stage 6 embryos with antisense oligodeoxynucleotide (ODN) complementary to FGF-2 mRNA were determined. Within 2 days of culture in defined medium, the explanted monolayer normally differentiates into a multilayer of proliferative cells that express sarcomeric α-actin and exhibit rhythmic contractions. The inclusion of 25 μM ODN that is complementary to the second exon of chicken FGF-2 mRNA caused a 50% inhibition in these cells' proliferative ability as judged by incorporation of 5′-bromodeoxyuridine; contractility was similarly inhibited. These effects were prevented by including recombinant human FGF-2 protein in the medium. Treatment with sense ODN did not cause inhibition. Inhibition of FGF-2 protein synthesis in the explanted tissue by antisense ODN was verified by immunoprecipitation analysis. These results point to a critical role for FGF-2 in the autocrine regulation of proliferation, and perhaps differentiative function, of embryonic cardiac myocytes.