It is well established that different protein classes undergo molecular evolution at different rates, presumably reflecting differing functional constraints. However, it is also the case that different isoforms of the "same" protein, encoded by a multigene family, may evolve at different rates. Here I report a relationship within gene families between isoform evolutionary rate and gene expression profile: Broadly expressed isoforms show stronger sequence conservation than do narrowly expressed isoforms. This observation emerged initially from cDNA cloning and sequencing studies, described here, of a vertebrate gene family encoding three differentially expressed isoforms of the muscle protein troponin I. However, the expression breadth/sequence conservation relationship applies to vertebrate gene families in general. In a broad and arbitrary survey sampling of sequence data on well-characterized vertebrate gene families, I found that in 14/15 families the most strongly conserved isoform was the most broadly expressed isoform, or one of several similarly broadly expressed isoforms. Broadly expressed isoforms are presumably subjected to greater negative selection pressure because they must function in a more diverse biochemical environment than do narrowly expressed isoforms. The expression breadth/evolutionary rate relationship has several interesting implications regarding the overall process of gene family evolution by duplication/divergence from ancestral genes.