Simple and efficient ex vivo/in vitro screening systems for contact allergens are developed for alternative to conventional animal tests. We have previously proposed an ex vivo/in vitro proliferation assay as a first stage screening method with advantages over existing alternatives, using lymph node cells (LNC) from sensitized guinea pigs of the Hartley strain. In this study, we have first confirmed, by histochemical analysis using in vivo bromodeoxyuridine (BrdU) and pyronin staining, that the ex vivo/in vitro LNC proliferation reflects in vivo response of lymph nodes to contact allergens. Furthermore, to improve the LNC assay, we then have investigated several experimental conditions for their influences on the LNC assay, demonstrating that, (1) the subscapular and the cervical LNC responded highly to contact allergens, (2) among three cervical lymph nodes the superficial dorsal cervical lymph nodes were the most reactive, (3) several vehicles alone used for animal sensitization exhibited little influence on the LNC proliferation, (4) employment of stimulation index offset the inter-batch fluctuation of the LNC proliferation in the control animals as baseline proliferation. Under optimized experimental conditions as above, experimentally determined stimulation indexes of several contact allergens correlated well with their sensitizing potential estimated by conventional animal tests. Therefore, the ex vivo/in vitro LNC proliferation assay should be a simple and efficient alternative to conventional guinea pig testings including the guinea pig maximization test (GPMT).