Relaxin is a member of the insulin-like growth factor family that functions primarily during pregnancy and parturition. Because previous studies have shown that rat adipocytes respond to relaxin, we undertook this study to investigate the effect of relaxin on the differentiation of 3T3-L1 fibroblasts in cell culture. First, relaxin prevented the normal course of cell division during the induction phase, but did not interfere with expression of the adipocyte phenotype, as indicated by lipid accumulation and insulin-sensitive glucose transport. Relaxin-treated cells were 2-3 times larger than cells induced by the normal procedure, as determined by both computer-assisted size analysis and intracellular water volume. These effects on cell division and cell size could be reversed by removal of relaxin during early induction. The concentration of relaxin required to elicit the half-maximal effect was 75 ng/ml (12.5 nM). These studies demonstrate that relaxin affects the proliferative response during the inductive phase of differentiation of the 3T3-L1 cell line without affecting differentiation directly. This effect of relaxin on proliferation is unique and is discussed in relation to the cell cycle. It is our hope that these cells will serve as a model system for investigating the mechanisms by which relaxin functions and allow further studies on receptor structure and function.