In response to the lack of a transgenic line of zebrafish labeled with heart‐specific fluorescence in vivo to serve as a research model, we cloned a 1.6‐kb polymerase chain reaction (PCR) ‐product containing the upstream sequence (−870 bp), exon 1 (39 bp), intron 1 (682 bp), and exon 2 (69 bp) of the zebrafish cardiac myosin light chain 2 gene, (cmlc2). A germ‐line transmitted zebrafish possessing a green fluorescent heart was generated by injecting this PCR product fused with the green fluorescent protein (GFP) gene with ends consisting of inverted terminal repeats of an adeno‐associated virus. Green fluorescence was intensively and specifically expressed in the myocardial cells located both around the heart chambers and the atrioventricular canal. Neither the epicardium nor the endocardium showed fluorescent signals. The GFP expression in the transgenic line faithfully recapitulated with the spatial and temporal expression of the endogenous cmlc2. Promoter analysis showed that the fragment consisting of nucleotides from −210 to 34 (−210/34) was sufficient to drive heart‐specific expression, with a −210/−73 motif as a basal promoter and a −210/−174 motif as an element involved in suppressing ectopic (nonheart) expression. Interestingly, a germ‐line of zebrafish whose GFP appeared ectopically in all muscle types (heart, skeletal, and smooth) was generated by injecting the fragment including a single nucleotide mutation from G to A at −119, evidence that A at −119 combined with neighboring nucleotides to create a consensus sequence for binding myocyte‐specific enhancer factor‐2. Developmental Dynamics 228:30–40, 2003. © 2003 Wiley‐Liss, Inc.