The establishment of precardiac mesoderm and the role of anterolateral endoderm and ectoderm in regulating heart muscle cell development have been studied in quail using explant cultures. Mesoderm from precardiac regions of stage 4+–6 embryos was explanted alone or in combination with adjacent endoderm or ectoderm, cultured for 12 to 72 hr in several types of culture media, and then assayed by morphological and immunocytochemical criteria for the presence of differnetiated cardiac myocytes. Results show that mesoderm from heart forming regions is capable of differentiating into beating cardiac myocytes in a defined medium lacking potential signaling molecules by stage 4+, the earliest time at which we could isolate mesoderm from adjacent cell layers. Although an interaction with anterolateral endoderm from stage 4+ onward is therefore not required for the specification of precardiac mesoderm in quail, explants consisting of mesoderm plus endoderm show an enhanced rate of myocyte differentiation and a shortened delay between expression of myosin heavy chain and the onset of beating. Endoderm also plays a central role in early heart morphogenesis since beating heart tubes from only in explants that contain both mesoderm and endoderm. In contrast, ectoderm from stage 4+–5+ embryos does not support development of precardiac mesoderm. These results suggeest that early heart muscle cell development involves an initial specification step that occurs prior to or during gastrulation and which leads to the appearance of myocardial precursor cells, and a subsequent differentiation step during which endoderm plays a central role in enhancing the rate of myocyte differentiation and the degree of heart tube morphogenesis. © 1994 Wiley-Liss, Inc.