Recent genome-wide analyses have revealed patterns of positive selection acting on protein-coding genes in humans and mammals. To assess whether the conclusions drawn from these analyses are valid for other vertebrates and to identify mammalian specificities, I have investigated the selective pressure acting on protein-coding genes of the puffer fishes Tetraodon and Takifugu. My results indicate that the strength of purifying selection in puffer fishes is similar to previous reports for murids but stronger in hominids, which have a smaller population size. Gene ontology analyses show that more than half of the biological processes targeted by positive selection in mammals are also targeted in puffer fishes, highlighting general patterns for vertebrates. Biological processes enriched with positively selected genes that are shared between mammals and fishes include immune and defense responses, signal transduction, regulation of transcription and several of their descendent terms. Mammalian-specific processes displaying an excess of positively selected genes are related to sensory perception and neurological processes. The comparative analyses also revealed that, for both mammals and fishes, genes encoding extracellular proteins are preferentially targeted by positive selection, indicating that adaptive evolution occurs more often in the extra-cellular environment rather than inside the cell. Moreover, I present here the first genome-wide characterization of neutrally-evolving regions of protein-coding genes. This analysis revealed an unexpectedly high proportion of genes containing both positively selected motifs and neutrally-evolving regions, uncovering a strong link between neutral evolution and positive selection. I speculate that neutrally-evolving regions are a major source of novelties screened by natural selection.