The induction of proliferation of adult rat islets was investigated under various culture conditions. The islets were isolated from male Sprague-Dawley rats and subsequently embedded in collagen gels, which mimic the in vivo three-dimensional surroundings. During the culture period, the effects of heterologous serum (fetal bovine serum, FBS), epidermal growth factor (EGF), and retinoic acid (RA) on islet growth were examined with respect to the morphological and total DNA content changes. To investigate these changes at the cellular level, whole mount immunocytochemistry using specific antibodies for insulin and glucagon was performed. The results showed that (i) collagen gels as an extracellular matrix can maintain islets in a similar way to that in vivo, (ii) heterologous serum (FBS) had stimulatory effects on islet proliferation in a dose-dependent manner, (iii) RA had inhibitory effects on islet proliferation induced by the serum in a dose-dependent manner, (iv) EGF had weak inhibitory effects on islet proliferation induced by the serum except at the concentration of 10 nM where its effect was not significant, and (v) whole mount immunocytochemistry revealed that newly proliferated islet cells were mainly $eta$-and $alpha$-cells.