In Saccharomyces cerevisiae, the histidine-containing phosphotransfer (HPt) protein YPD1 transfers phosphoryl groups between the three different response regulator domains of SLN1, SSK1, and SKN7 (designated R1, R2, and R3, respectively). Together these proteins form a branched histidine-aspartic acid phosphorelay system through which cells can respond to hyperosmotic and other environmental stresses. The in vivo order of phosphotransfer reactions is believed to proceed from SLN1-R1 to YPD1 and then subsequently to SSK1-R2 or SKN7-R3. The individual phosphoryl transfer reactions between YPD1 and the response regulator domains have been examined kinetically. A maximum forward rate constant of 29 s(-)(1) was determined for the reaction between SLN1-R1 approximately P and YPD1 with a K(d) of 1.4 microM for the SLN1-R1 approximately P.YPD1 complex. In the subsequent reactions, phosphotransfer from YPD1 to SSK1-R2 is very rapid (160 s(-)(1)) and is strongly favored over phosphotransfer to SKN7-R3. Phosphotransfer reactions between YPD1 and SLN1-R1 or SKN7-R3 were reversible. In contrast, no reverse transfer from SSK1-R2 approximately P to YPD1 was observed. These findings are consistent with the notion that SSK1 is constitutively phosphorylated under normal osmotic conditions. In addition, we have examined the roles of several conserved amino acid residues surrounding the phosphorylatable histidine (H64) of YPD1 using phosphoryl transfer reactions involving YPD1 mutants. With respect to phosphoryl transfer from SLN1-R1 approximately P, only one YPD1 mutant (K67A) exhibited an increase in K(d) and thus affects binding of YPD1 to SLN1-R1 approximately P, whereas other mutants (R90A, Q86A, and G68Q) showed a decrease in phosphoryl transfer rate. Only the G68Q-YPD1 mutant was significantly affected in phosphotransfer to SSK1-R2 ( approximately 680-fold decrease in rate in comparison to wild-type). This is the first report of a kinetic analysis of a eukaryotic "two-component" histidine-aspartic acid phosphotransfer system, enabling a comparison of the transfer rates and binding constants to the few bacterial systems that have been studied this way.