The RAG8 gene of Kluyveromyces lactis, which is one of the genes controlling the expression of the low-affinity carrier gene RAG1, has been cloned by in vivo complementation of the rag8 mutation. The sequence of Rag8p (535 amino acids), deduced from the nucleotide sequence of the cloned RAG8 gene, has been found to share a high degree of identity with the two casein kinases I of Saccharomyces cerevisiae, Yck1p and Yck2p, encoded by YCK1 and YCK2: the proteins are 65-66%, identical overall and show 89-90% identity in the kinase domain. The finding that the RAG8 gene of K. lactis cloned in a centromeric vector was able to complement the growth defect of a yck1 delta yck2(ts) mutant of S. cerevisiae strongly suggested that Rag8p is a casein kinase I. In contrast to the S. cerevisiae homologs, the RAG8 gene of K. lactis seems to be an essential single-copy gene, as shown by Southern blot experiments and the lethality of the rag8 null mutation. Northern blot analysis showed that the transcription of the RAG8 gene was higher on glucose media than in cells grown on a non-fermentable carbon source.