Using the vasopressin-sensitive rat mammary tumor cell line, WRK-1, we examined conditions under which vasopressin was able to stimulate turnover of prelabeled, radioactive phosphatidylinositol. Only cells which had been preincubated with 32Pi in the presence of hormone were able to subsequently respond to the hormone by increased loss of radioactivity from phosphatidylinositol. In addition, in experiments performed with uniformly labeled cells, we estimated that the hormone-sensitive phosphatidylinositol accounted for 17% of the total cellular phosphatidylinositol. Implications of these findings are discussed.