We have found that insulin-like growth factor binding protein-3 (IGFBP-3) accumulates to higher levels in medium conditioned by a strain of normal fibroblasts at late passage (LP) and a strain derived from subjects with Werner syndrome (WS) of premature aging, compared to medium conditioned by the same normal cells at early passage (EP) (Goldstein et al., Proc. Natl. Acad. Sci. USA, 88:9680-9684, 1991). To explore the generality of this phenomenon with respect to chronological age of donor (in vivo aging) and LP (in vitro senescence) we assayed IGFBP-3 in medium conditioned by 18 normal fibroblast strains at EP and LP and two WS strains at the midpoint of their curtailed replicative lifespans and assessed IGFBP-3 mRNA levels in cells by Northern analysis. The lowest accumulations of IGFBP-3 were found in medium conditioned by fetal cells with progressively increasing amounts postnatally; direct correlations between IGFBP-3 levels and donor age were seen in EP cells 3 days after subculture (during logarithmic growth) r = 0.80, P < 0.001, and 7 days after subculture (at confluence) r = 0.77, P < 0.001. With two exceptions, conditioned medium of cell strains accumulated more IGFBP-3 at LP; IGFBP-3 levels correlated with chronological age after 3 days, r = 0.50, P = 0.05, and after 7 days, r = 0.75, P < 0.001. IGFBP-3 content of WS culture medium fell within the range of LP normal cells. Cumulative IGFBP-3 levels were inversely proportional to the thymidine labeling index, a measure of proliferative vigor. With some exceptions IGFBP-3 mRNA levels were commensurate with the amount of IGFBP-3 accumulated in the medium, suggesting that distal translational and posttranslational mechanisms also regulate IGFBP-3 production in some strains. The trend toward augmented IGFBP-3 output of fibroblasts as a direct function of chronological age and in vitro senescence and as an inverse function of proliferative vigor is consistent with the known inhibitory effect of excess IGFBP-3 on IGF-mediated DNA synthesis and the reduced regenerative potential that is evident during biological aging in vivo.