The embryonic vertebrate heart is divided into two major chambers, an anterior ventricle and a posterior atrium. Although the fundamental differences between ventricular and atrial tissues are well documented, it is not known when and how cardiac anterior-posterior (A-P) patterning occurs. The expression patterns of two zebrafish cardiac myosin genes, cardiac myosin light chain 2 (cmlc2) and ventricular myosin heavy chain (vmhc), allow us to distinguish two populations of myocardial precursors at an early stage, well before the heart tube forms. These myocardial subpopulations, which may represent the ventricular and atrial precursors, are organized in a medial-lateral pattern within the precardiac mesoderm. Our examinations of cmlc2 and vmhc expression throughout the process of heart tube assembly indicate the important role of an intermediate structure, the cardiac cone, in the conversion of this early medial-lateral pattern into the A-P pattern of the heart tube. To gain insight into the genetic regulation of heart tube assembly and patterning, we examine cmlc2 and vmhc expression in several zebrafish mutants. Analyses of mutations that cause cardia bifida demonstrate that the achievement of a proper cardiac A-P pattern does not depend upon cardiac fusion. On the other hand, cardiac fusion does not ensure the proper A-P orientation of the ventricle and atrium, as demonstrated by the heart and soul mutation, which blocks cardiac cone morphogenesis. Finally, the pandora mutation interferes with the establishment of the early medial-lateral myocardial pattern. Altogether, these data suggest new models for the mechanisms that regulate the formation of a patterned heart tube and provide an important framework for future analyses of zebrafish mutants with defects in this process.