Polyamine uptake in Saccharomyces cerevisiae was modulated by extracellular magnesium; in a magnesium-limited medium, polyamine, especially spermine, was overaccumulated into the interior, whose level was then toxic for the growth of this organism (Maruyama, T., Masuda, N., Kakinuma, Y., and Igarashi K. (1994) Biochim. Biophys. Acta 1194, 289-295). Here we isolated a mutant (strain YTM22-8) whose growth was tolerant to spermine in magnesium-limited medium. This mutant was defective in polyamine uptake and did not overaccumulate spermine. From a yeast genome library we cloned a gene (POT1) which restored the spermine uptake of this mutant and the sensitivity of the growth to spermine. The nucleotide sequence of the POT1 gene indicated that it encodes a putative serine/threonine protein kinase and is located on chromosome XI. The results suggest that spermine uptake by this organism is probably regulated by phosphorylation and dephosphorylation.