The response of IMR-90 human fetal lung fibroblasts at high population doubling level (PDL > 42) to 1,25-dihydroxyvitamin D3[1,25(OH)2D3] was investigated to clarify whether some metabolic and molecular parameters of senescent cells are affected by the hormone treatment. Pyruvate kinase, lactate dehydrogenase and glucose-6-phosphate dehydrogenase activity significantly increased after treatment of confluent-phase cells with 10 nM 1,25(OH)2D3 for 24 h. Steroid specificity was established by the failure of 10 nM levels of 25-hydroxyvitamin D3 to affect the enzyme activities, while estradiol-17 beta and progesterone produced a slight increase in glucose-6-phosphate dehydrogenase and lactate dehydrogenase levels, respectively. 1,25(OH)2D3 also affected fibroblast proliferation, protein content/cell and DNA synthesis. The cell number significantly decreased after a 48 h incubation with 1,25(OH)2D3 at various concentrations (0.01-1 nM) when compared with control fibroblasts, while an increase in the protein content/cell was demonstrated. The same experiment, carried out by protracting the incubation with the hormone for 72 h, showed a similar trend, but 10 nM 1,25(OH)2D3 was also able to inhibit cell proliferation and to stimulate protein synthesis. The incorporation of [3H]thymidine into DNA increased after the treatment of high PDL fibroblasts with 0.01-1 nM of hormone for 48 h in comparison with controls.