Aim: To study the mechanisms of hepatitis B vaccine non-response, we examined hepatitis B virus surface antigen (HBsAg)-induced proliferation of peripheral blood mononuclear cells (PBMC) obtained from hepatitis B (HB) vaccinees. Methods: Subsequently, we have examined the features of HBsAg-reactive CD4+ T cells in HB vaccine non-responders (NR). Based on serum antibodies to HBsAg (anti-HBs) titers, we divided these vaccinees into three groups: high responder (HR), middle responder (MR) and non-responder (NR), and examined HBsAg-induced proliferation of their PBMC. Results: We found that the in vitro response of PBMC to stimulation with HBsAg was correlated with their serum anti-HBs titer (mean stimulation index was 10.71 in HR, 4.37 in MR and 1.96 in NR). However, by the deletion of CD8+ T cells, the increased response was observed in two of four NRs. Conclusions: The present results have also shown that at least four distinct HBsAg-reactive CD4+ clones existed (variable gene of T cell receptor beta (Vbeta) 17+ clone restricted with HLA-DR locus (DR4), Vbeta8+ clone restricted with HLA-DQ locust (DQ7), and both Vbeta5.1+ clone and Vbeta20+ clone restricted with either DR9 or DQ3) in NRs. The results demonstrated that heterogeneous HBsAg-reactive CD4 clones existed in some HB vaccine NRs. (C) 2001 Blackwell Science Asia Pty Ltd.