Insulin-like growth factor binding protein 3 (IGFBP-3) mRNA levels were consistently higher in both senescent normal human diploid fibroblasts (HDFs) at late passage (old cells) and prematurely senescent HDFs from a subject with Werner syndrome (WS) during serum depletion and repletion of growth medium and during proliferation from sparse to high-density inhibited cultures, compared to normal early-passage (young) HDFs. However, IGFBP-3 protein accumulated to higher levels in conditioned medium of old cells than in medium of WS and young cells, in that order, under the same conditions. Insulin-like growth factor I (IGF-I) was not detected in naive medium or in any of the media conditioned by these three cell types, whereas IGF-II was detectable in serum-repleted medium and remained relatively constant. Thus, molar ratios of IGFBP-3/IGF-II were consistently higher in old and WS cells and increased substantially as all three cell types became quiescent, due to either serum depletion or high cell density. These data are consistent with either an adaptive or a causal role for IGFBP-3 protein in the senescent and quiescent growth arrest of HDFs.