1α,25-dihydroxyvitamin D3 (1,25D3 ) is a highly potential anti-cancerous agent for prevention and treatment of prostate cancer, the most commonly diagnosed cancer type of males in western countries. A recent study by our laboratory, demonstrates that LNCaP cancer cells treated with 1,25D3, evoked dose-dependent activation of the JNK/SAPK MAPK signaling pathway within 10 minutes after hormone treatment, indicative of membrane-initiated steroid signaling (MISS) by 1,25D3. This confirms previous reports on intestinal-, chondrocyte- and osteoblast cells, where 1,25D3 operates through pharmacologically distinct nuclear-initiated mechanisms (NISS) and plasma membrane-initiated mechanisms. NISS is mediated via the vitamin D receptor (nVDR) and MISS is mediated through 1,25D3-MARRS (PDIA3, 1,25D3-membraneassociated rapid response steroid binding protein) or nVDR. The aims of the present study were to investigate the mechanisms of MISS evoked effects on alkaline phosphatase (ALP) and activation of the JNK/SAPK by 1,25D3, and the involvement of PDIA3 in 1,25D3 initiated activation of the JNK/SAPK signaling pathway. Furthermore, 1,25D3-treated LNCaP cells were transfected with siRNA against PDIA3 and phosphorylated JNK/SAPK was estimated by western analysis. Western analysis and ALP-assays demonstrated rapid activation of both JNK/SAPK as well as ALP. Silencing of PDIA3 did not affect 1,25D3 mediated activation of JNK/SAPK, suggesting that PDIA3 is not involved in the 1,25D3-initiated activation of the JNK/SAPK signaling pathway.