Little is known about the cis-acting elements controlling transcriptional activities of fish rhodopsin and myf-5 genes. Transgenic medaka and zebrafish were used to characterize promoters of carp (Cyprinus carpio) rhodopsin gene (cRh) and zebrafish (Danio rerio) myf-5 gene (myf-5), respectively. Transgenic medaka carrying different lengths of cRh upstream fragments fused with enhanced green fluorescence protein (EGFP) gene revealed several functional regions. Both upstream regions from -1261 to -163 bp (-1261/-163) and -163/-138 contributed to enhance the retina-specific activities of cRh. The cNRE (-75/-63) and CSE (-52/-46) motifs located at the cRh proximal promoter were sufficient to drive the transgene expressed in retinae. The -73/-68 within cNRE was also proved to be essential for the integrity of the minimal cis-regulatory elements. Regarding the myf-5, transgene expression in zebrafish embryos injected with different deletion fragments of zebrafish myf-5 upstream sequences showed that the -2776/-2456 and -1046/-844 regions contributed to the enhancer function. The -290/-154 region might be involved in controlling the translocation of progenitor muscle cells. Moreover, the upstream -82/-1 region was identified as a minimal promoter required for somite-specific expression. The -82/-62 region was the most critical sequence for myf-5 specificity.