Abstract Two functional properties of Leydig cells in culture i.e. LH-stimulated steroidogenesis and nuclear oestrogen receptor levels have been investigated. Leydig cells isolated from testes of immature rats and mature mice maintained their responsiveness to LH during 48–72 h of cell culture, although the mouse Leydig cells appeared to be less responsive to LH after 72 h of culture. In contrast, nuclear oestrogen receptor levels in both types of Leydig cells declined to 10–20% of the initial value after 24 h in culture. In the 48–72 h culture period nuclear oestrogen receptor levels recovered to 75% of the initial value only in Leydig cells from immature rats, whereas the nuclear oestrogen receptor levels in Leydig cells from mature mice remained low. These data demonstrate that during in vitro culture of Leydig cells, preservation of LH responsiveness does not necessarily warrant that other Leydig cell parameters e.g. nuclear oestrogen receptors also remain unaltered.