We have previously identified a novel population of small cells in human and canine pancreas characterized by immature morphology, quiescence, and a glucose-responsive insulin secretion. Based on their immature phenotype and predominant presence in small islets, we have hypothesized that small cells serve as islet progenitors. This hypothesis remains untested, however, due to persistent quiescence and scarcity of small cells in vitro. We have recently developed a culture medium that allowed for modest small cell proliferation. In this study we characterized the expression of genes potentially involved in small cell growth regulation by Q-RT-PCR. Our results suggest that quiescence of small cells correlates with up-regulation of Cdk inhibitors p27(Kip1), p16(INK4a) and p21(CIP1), PTEN, Hep27 and Foxo1a and with down-regulation of c-Myc and the receptors for EGF, FGF2 and HGF. The exit from quiescence correlates with activation of EGFR expression and down-regulation of p27(Kip1) and p16(INK4a). We also report here that small cells can be maintained in long-term non-adherent cultures preserving insulin and glucagon production for up to 208 days. Therefore, expansion of small cells in vitro may have a significant potential for the treatment of diabetes. This study is an important step in understanding the mechanisms involved in small cell growth regulation, which is required to fully evaluate their functional potential.