It is now recognized that significant tubular reabsorption of albumin occurs under physiological conditions that may play an important role in maintaining the proximal tubular integrity and function. Therefore, this study examined the effect of bovine serum albumin (BSA) on DNA synthesis and its related signal molecules in primary cultured rabbit renal proximal tubular cells (PTCs). BSA increased the level of [³H] thymidine incorporation in a dose (3 mg/ml) and time dependent manner (3 hr), [Ca²⁺]i and the level of protein kinase C (PKC) phosphorylation, and stimulated the phosphorylation of the epidermal growth factor receptor (EGFR), which was inhibited by EGTA (extracellular Ca²⁺ chelator), 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM, intracellular Ca²⁺ chelator), or PKC inhibitors (staurosporine or bisindolylmaleimide I). In addition, the PKC inhibitors or the EGFR inhibitor (AG1478) blocked the BSA-induced phosphorylation of p44/42 mitogen-activated protein kinases (MAPKs). BSA also increased the level of nuclear factor kappa B (NF-B) and inhibitor of NF-B (I-B) phosphorylation, which was blocked by staurosporine, AG1478, or PD98059 (p44/42 MAPK inhibitor). Inhibition of Ca²⁺, PKC, EGFR, p44/42 MAPKs, or NF-B signal pathways blocked the BSA-induced incorporation of [³H] thymidine. Consequently, the inhibition of Ca²⁺, PKC, EGFR, and p44/42 MAPKs, or NF-B blocked the BSA-induced increases in the cyclin D1, cyclin dependent kinase 4 (CDK4), cyclin E, or CDK2 and restored the BSA-induced inhibition of the p21^WAF/Cip1 and p27^Kip1 expression. In conclusion, BSA stimulates DNA synthesis that is mediated by Ca²⁺/PKC as well as the EGFR-dependent p44/42 MAPKs and NF-B signal pathways in PTCs.