The Saccharomyces cerevisiae cell cycle is arrested in G(1) phase by the mating factor pathway. Genetic evidence has suggested that the G1 cyclins Cln1, Cln2, and Cln3 are targets of this pathway whose inhibition results in G(1) arrest, Inhibition of Cln1- and Cln2-associated kinase activity by the mating factor pathway acting through Far1 has been described, Here we report that Cln3-associated kinase activity is inhibited by mating factor treatment, with dose response and timing consistent with involvement in cell cycle arrest, No regulation of Cln3-associated kinase was observed in a fus3 kss1 strain deficient in mating factor pathway mitogen-activated protein (MAP) kinases, Inhibition occurs mainly at the level of specific activity of Cln3-Cdc28 complexes, Inhibition of the C-terminally truncated Cln3-1-associated kinase is not observed; such truncations were previously identified genetically as causing resistance to mating factor-induced cell cycle arrest, Regulation of Cln3-associated kinase specific activity by mating factor treatment requires Farl, Overexpression of Farl restores inhibition of C-terminally truncated Cln3-1-associated kinase activity, G(2)/M-arrested cells are unable to regulate Cln3-associated kinase, possibly because of cell cycle regulation of Farl abundance, Inhibition of Cln3-associated kinase activity by the mating factor pathway may allow this pathway to block the earliest step in normal cell cycle initiation, since Cln3 functions as the most upstream G(1)-acting cyclin, activating transcription of the G(1) cyclins CLN1 and CLN2 as well as of the S-phase cyclins CLB5 and CLB6.