SUMMARY: The occurrence of choline sulphate in 32 fungi and 9 bacteria was investigated by incorporating Na235SO4 into culture media and subsequently identifying radioactive choline sulphate in cell extracts by chromatography and radioautography. The production of choline sulphate appears to be restricted to higher fungi since it was not found in any of the tested Phycomycetes. Of the Ascomycetes, the Endomycetales did not synthesize choline sulphate but the mycelia of the Sphaeriales did accumulate the compound. The Basidiomycetes and all the Fungi Imperfecti, except Torulopsis utilis, were choline sulphate positive. The choline sulphate negative organisms, including all of the bacteria tested, could not be induced to produce choline sulphate by adding large amounts of choline to the culture media or by growing on chemically defined media in which inorganic sulphate was the sole sulphur source. On the other hand, Aspergillus oryzae produced choline sulphate on a minimal medium containing inorganic sulphate, cysteine, cysteic acid or taurine as sulphur sources. The distribution of choline sulphate production has been discussed in terms of the absence or presence of the enzyme choline sulphokinase.