In rats, during postnatal Leydig cell development, the progenitor Leydig cells (PLC) proliferate actively during days 14-21 of postnatal life. Luteinizing hormone (LH) is known to stimulate Leydig cell proliferation and oestradiol 17β inhibits this process. In order to identify the molecules involved in Leydig cell proliferation, differentially expressed genes in proliferating and non-proliferating PLC isolated from vehicle and oestradiol 17β-treated rats respectively, were analysed by differential display reverse transcription polymerase chain reaction (DD-RT-PCR). Results revealed that the expression of collagen IV α4 (Col IV α4), a subunit of extracellular matrix (ECM) protein collagen IV, was down regulated in PLC isolated from oestradiol 17β-treated rats. Studies on stage specific expression of Col IV α4 during Leydig cell development revealed that this transcript is abundantly expressed at the stage where Leydig cell proliferation is maximal and the expression of this transcript decreased during differentiation of Leydig cells, which is associated with loss of proliferation. These observations suggest that Col IV α4 is important for PLC proliferation. Stimulation of PLC proliferation in vitro in the presence collagen IV provides additional support for the conclusion that collagen IV-mediated signalling is involved in PLC proliferation. Further studies revealed that active forms of focal adhesion kinase (FAK) and mitogen activated protein kinase 1/2 (MAPK 1/2), the intracellular signalling molecules that are known to mediate ECM protein signalling are present only in proliferating forms of Leydig cells and are absent in non-proliferating Leydig cells. These results suggest that collagen IV-mediated signalling is involved in PLC proliferation.