This study was undertaken in order to analyse the immuno-regulatory mechanism of non-responsiveness to hepatitis B (HB) vaccine in healthy individuals. We have reported already that peripheral mononuclear cells (MNC) isolated from anti-HBs sero-positive subjects after booster injection are able to make anti-HBs in vitro by stimulation with pokeweed mitogen (PWM) plus HBsAg. In contrast, under the same conditions, non-responder (NR) MNC failed to produce anti-HBs in vitro, even after an additional injection, although an amount of immunoglobulin was synthesized corresponding with the responder controls. Co-culture experiments carried out with T-cell fractions (T) and non-T-cell fractions (NT) from NR and controls showed: (i) by Week 2 after the last vaccination, NT from NR could hardly be activated to produce anti-HBs; however, by Week 4 significant synthesis of anti-HBs was induced by the stimulation of responder T; (ii) in experiments using responder NT and T from NR, seven out of eight and five out of nine co-cultures were found to make anti-HBs by Weeks 2 and 4, respectively; (iii) T from NR, as well as their culture supernatants, specifically suppressed anti-HBs production of responder MNC, especially by Week 4 after the last vaccination. From these results, it is predicted that the early stage of non-responsiveness in NR is mainly due to a defect of the B-cell repertoire, while that of the late stage is caused by the existence of HBsAg-specific suppressor T cells.