Key factors which impact on the biosynthesis and subsequent fate of the phospholipid precursor inositol were studied as a function of growth phase in the yeast Saccharomyces cerevisiae. Both wild-type and strains disrupted for the OPI1 gene, the principal negative regulator of the phospholipid biosynthetic genes, were examined. Overexpression of the INO1 gene and overproduction of both inositol and the major inositol-containing phospholipid, phosphatidylinositol, varied as a function of growth phase. In opi1 cells, INO1 expression was constitutive at a high level throughout growth, although the level of transcript was reduced at stationary phase when the cells were grown in defined medium. In the wild-type strain, INO1 expression was limited to a peak in the exponential phase of growth in cells grown in the absence of inositol. Interestingly, the pattern of OPI1 expression in the wild-type strain resembled that of its putative target, INO1. Intracellular inositol contents of the opi1 strain were higher than those of the wild-type strain, with peak levels occurring in the stationary phase. Membrane phosphatidylinositol content paralleled intracellular inositol content, with opi1 strains having a higher phosphatidylinositol content in stationary phase. The proportion of the predominant phospholipid, phosphatidylcholine, exhibited a profile that was the inverse of the phosphatidylinositol content: phosphatidylcholine content was lowest in opi1 cells in stationary phase. The opi1 mutation was also found to have effects beyond phospholipid biosynthesis. opi1 cells were smaller, and opi1 cultures achieved a cell density twice as high as comparable wild-type cultures. opi1 cells were also more salt tolerant than wild-type cells: they were partly resistant to shrinking, more rapidly resumed growth, and attained a higher culture density after upshift to medium supplemented with 8% NaCl.