Muscle mitochondrial content varies widely among fiber-types and species. We investigated the origins of variation in the activity of the mitochondrial enzyme citrate synthase (CS), an index of mitochondrial abundance, among fiber-types and species of high performance fish (tunas and billfishes). CS activities varied up to 30-fold among muscles: lowest in billfish white muscle and highest in billfish heater organ. Among species, CS activities of red, white and cardiac muscle of 3 tuna species were 2-fold greater than the homologous muscles of 2 billfish species. Since comparisons of CS amino acid sequences deduced from a combination of PCR methods argue against clade-specific differences in catalytic properties, CS activity reflects CS content among these 5 species. To assess the basis of these differences in CS activity we looked at the relationship between CS activity (U/g muscle), nuclear content (DNA/g muscle), and CS transcript levels (CS mRNA/g RNA). Muscle CS activity differed by 10- to 30-fold when expressed per g muscle, but only 3-fold when expressed per mg DNA. Thus, it is nuclear DNA content, not fiber-type differences in CS gene expression that may be the main determinant of CS activity in muscle. Conversely, evolutionary (tunas vs. billfishes) differences in CS arise from differences in posttranscriptional regulation, based upon relationships between CS enzyme levels and CS mRNA assessed by quantitative competitive RT-PCR. These data argue that fiber-type differences can arise without major differences in fiber-type specific regulation of the CS gene, whereas evolutionary differences may be largely due to post-transcriptional regulation.