Abstract Thermal inactivation of hepatitis B surface antigen (HBs Ag) was investigated under various experimental conditions by means of a quantitative radioimmunoassay. No decrease of HBs Ag immunologic reactivity could be detected at -80°C, up to 24 months of storage, whereas at -20 and 4°C survival of serum HBs Ag after 6 months was 71.4 and 48.7%, respectively. Freezing and thawing of serum affected the activity of the antigen slightly (60.6% after 26 cycles in 6 months). The loss of activity of purified HBs Ag in phosphate buffer at 20, 37, 44, 56, 70, and 98°C was linear with respect to time, and the half-life of inactivation ranged from 6 min at 98°C to 52 days, 18 hr, and 12 min at 20°C. Over this range of temperature, the velocity constants for inactivation (k) showed a linear relationship with the reciprocal of absolute temperature, and the energy (ΔH) and the entropy (ΔS) of activation of the process were 23.382 kcal/mole and -8.468 cal/mole/deg, respectively. No residual activity was detected after 15 min at 121°C (1 Atm). At neutral pH an increasing protective effect was afforded by deionized water, 0.15 M NaCl, 0.067 phosphate mixture, 0.1 M citric acid-0.2 M sodium phosphate dibasic buffer and human serum. Heat-stability of HBs Ag was significantly reduced by modifying the pH of the medium whereas addition of 1 M MgCl2 had a considerable stabilizing effect. Survival of HBs Ag was affected by contamination with a variety of microbial strains. Traces of the antigen dried on a glass surface could be recovered after 3 and 4 weeks of exposure at room temperature (21 ± 2°C) to ambient air. Subtypes adw and ayw were destroyed to the same extent by heat exposure, and no selective inactivation of a, d, and y subdeterminants was observed.