Alternative splicing of pre-messenger RNA (pre-mRNA) is a highly regulated process that allows expansion of the potential of expression of the genome in higher eukaryotes and involves many factors. Among them, the family of the serine- and arginine-rich proteins (SR proteins) plays a pivotal role: it has essential functions during spliceosome assembly and also interacts with RNA regulatory sequences on the pre-mRNA as well as with multiple cofactors. Collectively, SR proteins, because of their capacity to recognize multiple RNA sequences with a broad specificity, are at the heart of the regulation pathways that lead to the choice of alternative splice sites. Moreover, a growing body of evidence shows that the mechanisms of splicing regulation are not limited to the basic involvement of cis- and trans-acting factors at the pre-mRNA level, but result from intricate pathways, initiated sometimes by stimuli that are external to the cell and integrate SR proteins (and other factors) within an extremely sophisticated network of molecular machines associated with one another. This review focuses on the molecular aspects of the functions of SR proteins. In particular, we discuss the different ways in which SR proteins manage to achieve a high level of specificity in splicing regulation, even though they are also involved in the constitutive reaction.