The predominant integral membrane protein of the CNS myelin of amphibia, reptiles, birds and mammals is proteolipid protein (PLP) and P0, the main glycoprotein in PNS myelin. Alternative splicing of the transcripts of the single genes of PLP and myelin basic protein (MBP) is the underlying mechanism by which the isoforms of the two main proteins of the myelin membrane arise. DM20 is an isoform of PLP in mammalian, avian and reptilian myelin. It does not occur in the CNS myelin of amphibia. DM20 lacks an extended hydrophilic sequence exposed on the extracytoplasmic surface of the lipid bilayer as a result of the usage of a cryptic donor splice site within exon III. We report about comparative studies on PLP and its DM20 isoform on the protein and DNA level of frog, chicken, rat CNS and the P0-related IP proteins of the CNS of trout. Chemical cleavage at tryptophan residues with N-chlorosuccinimide yields identical patterns of PLP peptides which refers to a high conservation between amphibia, birds and mammals and is totally different from the cleavage pattern of hydrophobic myelin proteins IP-1 and IP-2 of trout CNS and that of P0 of rat PNS. The N-terminal 19 amino-acid residues of IP-1 of trout CNS- and P0 of frog PNS myelin were sequenced and proved to be homologous on one hand with the P0 analogue of CNS of the shark, a cartilage fish, and on the other hand with P0 protein of PNS of birds and mammals. The complete amino-acid sequence of chicken CNS PLP was derived from its cDNA. Coding and noncoding segments of the PLP gene of frog were sequenced: there is a high degree of conservation between amphibian and mammalian PLP within the hydrophobic domains. Numerous mutations were found within the part of exon III encoding the hydrophilic domain. Base exchanges within the putative splice site in exon III explain the absence of DM20 in the protein pattern of amphibia CNS myelin. This result is being discussed in view of the membrane organization and the function of PLP.