A variety of lower eukaryotes and certain mammalian cells are known to constitutively secrete lysosomal hydrolases. Recent studies in Tetrahymena have shown that phosphatidylinositol 3-phosphate regulates the proper secretion of lysosomal enzymes at the level of phagolysosome formation. We extend these findings by studying the secretion-deficient strain MS-1 of Tetrahymena thermophila, which possess phosphatidylinositol levels similar to wild type. However, steady-state levels of phosphatidylinositol 4-phosphate (PtdIns4P) were found to be doubled in this strain compared with wild type as shown by in vivo [3H]inositol labeling and high-performance liquid chromatography analysis. The increased PtdIns4P levels in MS-1 cells were unrelated to the upregulation of total phosphatidylinositol phosphate induced by hyperosmotic stress because this treatment resulted in a similar increase of PtdIns4P in MS-1 and wild-type cells. Hyperosmotic stress did not affect secretion in either of the two types of cells. On the other hand, under conditions of wortmannin-induced hypersecretion in wild-type cells, MS-1 cells developed a highly vacuolated phenotype while secretion was not induced. Importantly, comparative analysis of wild-type and MS-1 cells under wortmannin treatment showed that PtdIns4P levels were differentially regulated in the two strains. These results collectively suggest that PtdIns4P turnover in Tetrahymena is linked to lysosome secretion.