We recently identified a novel calcium-regulated gene, HCaRG, that is highly expressed in the kidney and maps to a chromosomal locus determining kidney weight in rats. The mRNA levels of HCaRG negatively correlate with the proliferative status of the kidney cells. To investigate its role in renal epithelial cellular growth directly, we studied the human embryonic kidney cell line (HEK-293) stably transfected with either plasmid alone or plasmid containing rat HCaRG. [³H]thymidine incorporation was significantly lower in HCaRG clones. Although HCaRG clones exhibited some enhanced susceptibility to cell death, this was not the primary mechanism of reduced proliferation. Cell cycle analysis revealed a G₂M phase accumulation in HCaRG clones that was associated with upregulation of p21^Cip1/WAF1 and downregulation of p27^Kip1. HCaRG clones had a greater protein content, larger cell size, and released 4.5- to 8-fold more of an atrial natriuretic peptide-like immunoreactivity compared with controls. In addition, HCaRG clones demonstrated the presence of differentiated junctions and a lower incidence of mitotic figures. Genistein treatment of wild-type HEK-293 cells mimicked several phenotypic characteristics associated with HCaRG overexpresssion, including increased cell size and increased release of atrial natriuretic peptide. Taken together, our results suggest that HCaRG is a regulator of renal epithelial cell growth and differentiation causing G₂M cell cycle arrest.