Cytokinins are a class of naturally occurring compounds that regulate growth and differentiation in tissues of higher plants. Many cytokinins are isopentenylated derivatives of adenine and its riboside, adenosine. By virtue of the post-transcriptional isopentenylation of specific anticodon loop adenosine residues in certain tRNA sequences, cytokinins are nearly universal, but tRNA-independent (de novo) cytokinin synthesis has been demonstrated in a few species. Using a radioimmunoassay, we have demonstrated that haploid strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe contain, respectively, 0.8 and 0.9 microgram of the free cytokinin, N6-(delta 2-isopentenyl)adenosine, per g of cells (wet weight). Strains of both species characterized by mutations that result in deficiencies of isopentenylated tRNAs have somewhat elevated levels of free N6-(delta 2-isopentenyl)adenosine. These findings lead to the conclusion that the major, if not exclusive, source of free cytokinins in these two yeasts is a synthetic pathway independent of isopentenylated RNA turnover.