We examined the activity of the bone morphogenetic protein 4 (BMP4) promoter in zebrafish embryos via transient and stable transgenic expression analyses in order to obtain a better understanding of the regulation of BMP4 tissue-specific expression. Transient expression studies showed that the 9.0-kb BMP4 promoter/upstream region drove green fluorescent protein (GFP) expression mainly in the heart. Deletion analyses indicated the existence of multiple regulatory elements in the 7.5-kb BMP4 promoter/proximal upstream region. In addition, a coinjection experiment further demonstrated the 2.4-kb Bgl II-Hind III DNA region contains major positive regulatory elements. In addition, stable transgenic lines were established to further confirm the heart-specificity of this segment in BMP4 promoter. The results showed that GFP was mainly localized in the myocardium of developing ventricles of 48-hpf (hours postfertilization), 72-hpf, and 100-hpf transgenic F1 embryos. Together, these results indicate that the 7.5-kb BMP4 promoter/proximal upstream region specifically contains regulatory elements for BMP4 expression in the heart, while regulatory elements for other endogenous BMP4-expressing tissues may reside in more distal regions and/or in introns.