Nutritional folate deficiency in Chinese hamster ovary (CHO)-K1 cells inhibited population growth rate and caused growth arrest within 3 days of culture in Fol- medium [without folate, hypoxanthine (Hx), and thymidine (TdR)]. Coincident with impaired population growth was a transient delay in cell cycle progression through S phase and an increase in cell size. The growth-arrested population of predominantly G1 phase cells exhibited an increased adhesion to the culture substratum. There was a time-dependent loss of cell reproductive capacity. All these various perturbations of cellular phenotype induced by folate deficiency were prevented by the addition of folate or a combination of TdR and Hx to the Fol- medium. However, the singular presence of each nucleotide precursor differentially affected the pleiotropic response. The addition of Hx to Fol- medium exacerbated the aforementioned abnormalities, producing a threefold increase in mean cell volume, a 72 hr accumulation of cells in the S phase of the cell cycle, and a rapid demise in cell clonogenicity. Unexpectedly, we found reduced cell adhesion in these cultures. In contrast, folate-deficient cells supplemented with TdR exhibited a general amelioration of cell perturbations with respect to cell size, cell cycle distribution, and reproductive viability. Notably, such populations were not released from growth inhibition or subsequent growth arrest, and the cells became elongated and highly adherent with time. When cell populations from each of the three conditions of folate-deficient culture were released from growth arrest by addition of complete medium, the respective profiles of synchronous cell cycle progression were distinctive.