Pseudomonas C(12)B and other Pseudomonas species released larger amounts of a (35)S-labelled metabolite into the medium when cultured on growth-limiting concentrations of Na(2)SO(4) as opposed to growth in SO(4) (2-)-sufficient media. The metabolite was found at all stages of the culture cycle of Pseudomonas C(12)B and maximum quantities occurred in stationary-phase culture supernatants. The metabolite was not detected when the bacterium was cultured on growth-limiting concentrations of potassium phosphate. The amount of the metabolite present in the medium greatly exceeded that which could be extracted from intact cells and, except for choline chloride, it was independent of the carbon source used for growth. If choline chloride was present in high concentration, then larger amounts of the metabolite were found in the culture medium. The metabolite was not detected extracellularly or intracellularly when the bacterium was grown in SO(4) (2-)-deficient media containing 5mm-l-cysteine. The same metabolite was also synthesized in vitro only when Pseudomonas C(12)B extracts were incubated with choline chloride, ATP, MgCl(2) and Na(2) (35)SO(4). The metabolite-forming system was not subject to repression by Na(2)SO(4) and was completely inhibited by 0.5mm-l-cysteine and activated by Na(2)SO(4) (up to 1.0mm). The metabolite was identified as choline O-sulphate by electrophoresis, chromatography and isotope-dilution analysis. Another (35)S-labelled metabolite was also detected in culture supernatants, but was not identified.