Recently, we have isolated a Chinese hamster cell variant (IV-A1 -j) resistant to an insulin-diphtheria-A chain toxic conjugate (Leckett and Germinario: Cytotechnology [in press]. This cell line exhibited a decreased level of insulin binding, but normal growth in serum-containing medium when compared to the parental cell line (V-79). In this paper we further demonstrate that while IV-Al-j cells are capable of growing in serum-containing medium, they are insensitive to the mitogenic actions of either insulin or IGF-1. In contrast, epidermal growth factor (EGF) and/or a-thrombin (THR) generate a mitogenic effect in IV-A1 -j cells comparable to that observed in the parental V-79 cells. The combination of EGF and/or THR with either insulin or IGF-l results in an increase in V-79 cell growth above EGF and/or THR alone. On the other hand, insulin or IGF-1 in the presence of other mitogens did not stimulate further growth in IV-A1-j cells. While insulin binding was lower in IV-A1-j cells, internalization of '"I-insulin was not different in the two cell types. Additionally, insulin-stimulated glycogen synthesis and protein synthesis were not different in the two cell types. These observations are consistent with insulin and IGF-1 sharing a mitogenic signalling pathway in Chinese hamster fibroblasts and that this pathway is distinct from other growth factor signalling pathways. The fact that this pathway is defective in the IV-A1-j cell line indicates t h e potential usefulness of these cells in identifying a key step(s) in the insulin (IGF-1) mitogenic pathway. o 1993 Wiley-Liss, Inc.