We examined the mutagenicity of iso-butyl nitrite (IBN) vapor and aqueous IBN solution in the Ames test to help evaluate the hazard of sniffing this vapor, a habit which might play a role in the induction of Kaposi's sarcoma associated with acquired immune deficiency syndrome. Chemical analysis showed that the saturated vapor contained 190 micrograms IBN/ml at 25 degrees C, and saturated aqueous solution, 2.6 mg IBN/ml at 21-23 degrees C. When agar plates containing Salmonella typhimurium TA-1535 and rat liver S-9 were exposed to IBN vapor, the number of mutants reached a maximum after 40 min. A mean of 307 mutants/plate (22 x background) was observed when the plates were exposed to IBN vapor for 30 min. Addition of 0.2 ml saturated IBN solution in water to similar plates gave a mean of 179 mutants/plate (7.9 x background) in the absence of S-9, confirming published results. The S-9 did not affect the results. Based on the IBN level in medium exposed to IBN vapor, the vapor was apparently 11 times more mutagenic than IBN solution. This was attributed to continuous replenishment of unstable IBN in the medium by the vapor. The half-life of IBN at 21-23 degrees C was > 1 hr for solutions in water and < 3 min for solutions in the assay medium. This instability was traced to a reaction with phosphate, presumably hydrolysis to nitrite and iso-butanol. IBN in solution was 2.8 times more mutagenic than sodium nitrite, suggesting that IBN was not mutagenic because of its conversion to nitrite. Iso-butanol was not mutagenic. The results demonstrate the potential hazard of sniffing IBN vapor.