We have investigated the change in DNA supercoiling in Bacillus subtilis after exposure to different kinds of stress. No or only minor effects are induced by solvents. Anaerobiosis and heat shock result in plasmids which are less negatively supercoiled. Cold shock and salt stress result in an increase in negative supercoiling. The reduction of linking number starts immediately after addition of NaCl, and reached a maximal value after 2 min. Primer extension experiments revealed that the mRNA level of the gyrA gene is not specifically changed by stress. The response in B. subtilis is inverse to the response in vitro. We discuss the possible effects of these inverse in vivo and in vitro responses for B. subtilis.