Insulin-like growth factor-I (IGF.I) stimulation of basal keratinocytes is an essential component of normal epidermal homeostasis. In addition to the IGF receptor, basal keratinocytes synthesize insulin-like growth factor binding protein-3 (IGFBP-3). The HaCaT keratinocyte cell line, which has many characteristics of basal keratinocytes, synthesizes IGFBPI73 that in vitro reduces its IGF-I responsiveness. IGFBP-3 has attracted interest as a potential growth arrest protein, both via its ability to modulate IGF-I responsiveness, and more controversially via IGF-I-independent mechanisIias. Intracellular modes of action have been proposed, and a nuclear localization consensus sequence has previously been identified within IGFBP-3. Using immunocytochemistry with a biotinylated antibody specific for IGFBP-3, we investigated the intracellular localization of IGFBP-3 in subconfluent monolayer cultures of HaCaT cells. Diffuse cellular staining was visible, potentially corresponding to cell surface and nascent cytoplasmic IGFBP-3. Of particular interest however, was the localization of staining over the nuclei of a large proportion of cells that were undergoing cell division. Antibody staining was specific for IGFBP-3 because addition of recombinant human IGFBP-3 to the antibody prior to incubation with the ceils inhibited these staining patterns. Optical sections obtained using a confocal laser scanning microscope showed that in keratinocytes undergoing cell division, IGFBP-3 was localized inside the nucleus. These results show that intracellular IGFBP-3 localization is altered during the cell cycle and suggest a possible nuclear role for IGFBP-3 during cell division.