Creatine kinase (CK) catalyzes the reversible transfer of thegamma-terminal phosphate of MgATP to the guanidine creatine (Cr) forming MgADP and phosphocreatine (PCr). The CK reaction plays a central role in both temporal and spatial ATP buffering in cells displaying high and variable rates of ATP turnover. There is a constant non-enzymatic conversion of Cr and PCr to creatinine that must be compensated for by biosynthesis and/or dietary uptake. In all true vertebrate craniates, there is a capacity for de novo biosynthesis of Cr as evidenced by the presence of the two enzymes involved in the biosynthetic pathway-arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT). Many protochordates and a broad spectrum of invertebrates, including the basal metazoan sponges, express CK and contain significant pools of Cr/PCr, particularly in primitive-type spermatozoa. However, attempts at demonstrating the enzymes of Cr biosynthesis in these organisms have failed and it has been suggested that Cr is derived from the diet and/or by direct uptake from seawater. We show in this communication that the protochordate tunicate Ciona intestinalis expresses GAMT based on the deduced amino acid sequence of a cDNA amplified by reverse transcription PCR. To validate that the transcript coded for GAMT, a full length cDNA was generated by PCR amplification and ligated into an expression vector. The resulting recombinant protein had an N-terminal amino acid sequence and relative molecular mass as predicted by the deduced amino acid sequence from the cDNA. Catalytic studies of this recombinant GAMT showed that it indeed had the capacity to methylate guanidinocetate to Cr with an apparent K(m) and maximal velocity comparable to GAMTs from vertebrates. Real time PCR showed that this GAMT is primarily expressed in the stomach and gonad, in close proximity to where Cr is packaged into spermatozoa, but also is expressed in two other tissue complexes. Analysis of the C. intestinalis genome and EST sequencing projects showed that the AGAT gene is present and is expressed demonstrating that this species has the complete Cr biosynthetic pathway. Perusal of other EST and genome sequencing projects revealed that true GAMTs are present in the lancelet Branchiostoma, the sea urchin Strongylocentrotus and the hydroid Hydractinia and AGAT genes are present in both Branchiostoma and Strongylocentrotus. Given our present experimental results and the emerging EST/genome sequencing data, it is clear that the capacity for de novo Cr biosynthesis is likely widespread in protochordates and invertebrates expressing CK and that the genes for GAMT/AGAT likely evolved coincident with CK.