We have isolated a new gene (PTK2) which restores spermine uptake of a polyamine uptake-deficient mutant of Saccharomyces cerevisiae (Kakinuma, Y., Maruyama, T., Nozaki, T., Wada, Y., Oshumi, Y., and Igarashi, K., 1995, Biochem, Biophys. Res. Commun. 216, 985-992). In magnesium-limited medium, the cell growth of a spermine-sensitive polyamine uptake mutant transformed with PTK2 recovered its sensitivity to spermine. The nucleotide sequence of the PTK2 gene indicated that it is identical with the YJR059W open reading frame of chromosome X encoding a putative serine/threonine protein kinase. The deduced amino acid sequence of the PTK2 gene product was 38% identical and 55% similar with that of the PTK1 (POT1) gene product, a putative serine/threonine protein kinase, which was found to enhance spermine uptake of the same mutant. The results indicate that polyamine transport of yeast is regulated by multiple phosphorylation/dephosphorylation pathways.