To study the process of ventricular specification during cardiogenesis, we examined the in situ expression of cardiac ventricular myosin light chain 2 (MLC-2v) mRNA during murine embryogenesis. As assessed by hybridization with a specific MLC-2v riboprobe, mRNA expression can be found in the ventricular region at day 8.0 postcoitum (pc). MLC-2v expression is high in the ventricular portion of the heart tube, with no detectable expression in the atrial or sinus venosus regions. The proximal outflow tract of the heart tube also expresses MLC-2v mRNA at minimally detectable levels at this time but then displays a temporally and spatially distinct pattern with expression well established in the proximal out-flow tract region adjacent to the ventricular segment by days 9-10 pc, eventually reaching levels comparable to the trabeculated ventricular myocardium. By day 11 pc, prior to the completion of septation, expression then becomes restricted to the ventricular region at and below the level of the atrioventricular cushion. Transgenic mice harboring a 250-base-pair MLC-2v promoter fragment fused to a luciferase reporter gene demonstrate reporter gene activity from at least day 9 pc. Ventricular region-restricted expression of the luciferase reporter in the embryonic heart, as assessed by immunofluorescence and direct assay of reporter activity in microdissected atrial and ventricular muscle specimens, was confirmed from at least day 15 pc on. Taken together, this provides evidence for early positional specification of MLC-2v gene expression in the primitive heart tube and indicates regional specification of part of the ventricular muscle gene program can precede ventricular septation during mammalian cardiogenesis. Since the 250-base-pair promoter fragment is active developmentally in transgenic mice, this establishes it as a molecular target for the process of ventricular specification in the developing heart tube.