The cyclic purine nucleotides cAMP and cGMP are well-established second messenger molecules that are generated by distinct nucleotidyl cyclases (NCs) and regulate numerous cell functions via specific effector molecules. In contrast, the existence of the cyclic pyrimidine nucleotides cCMP and cUMP has been controversial for many years. The development of highly specific and sensitive mass spectrometry methods has enabled the unequivocal detection and quantitation of cCMP and cUMP in biological systems. These cNMPs occur broadly in numerous mammalian cell lines and primary cells. cCMP has also been detected in mouse organs, and both cCMP and cUMP occur in various developmental stages of the zebrafish Danio rerio. So far, the soluble guanylyl cyclase (sGC) and soluble adenylyl cyclase (sAC) have been identified as cCMP and cUMP generators. Dissociations in the expression patterns of sAC and sGC relative to cCMP and cUMP abundance may point to the existence of hitherto unidentified cCMP- and cUMP-generating NCs. The broad occurrence of cCMP and cUMP in vertebrates and the distinct cNMP patterns suggest specific roles of these cNMPs in the regulation of numerous cell functions.