Elemental sulphur (32S0 detected as 32S8) is the only inorganic phytoalexin recorded, first identified as a component of resistance of Theobroma cacao to Verticillium dahliae. Here we report S0 production in another four, diverse families in response to fungal and bacterial pathogens. S0 was detected in xylem excised from infected but not control tomato and cotton to V. dahliae, tobacco and French bean to Fusarium oxysporum and tomato to Ralstonia solanacearum. Accumulation was more rapid and greater in disease resistant genotypes than in susceptible lines. No S0 was detected in strawberry challenged with V. dahliae or maize leaves with Erwinia stewartii. Furthermore, S0 was absent from leaves of six spp. undergoing hypersensitive reactions to Pseudomonas syringae pathovars. These data suggest that S0 production may be specific to xylem and does not occur in all plants. Anomalously, high constitutive levels of S0 occurred in control and hypersensitively responding leaves of Brassica oleracea, irrespective of challenge by an incompatible race of Peronospora parasitica and in uninoculated Arabidopsis thaliana leaves. Scanning electron microscopy–energy dispersive X-ray microanalysis showed sulphur in A. thaliana was present in all leaf cell types. S8 showed high toxicity (ED50 0.78–3.125 μg ml−1 to spore germination and germ tube growth) to isolates of V. dahliae and F. oxysporum but had no effect on bacterial pathogens or to a Phytophthora sp. Levels in tomato (10 μg g−1) and Arabidopsis (1.5–6 μg g−1) were potentially inhibitory, but in other spp. (maximum 100–250 ng g−1) were below theoretical inhibitory levels. The highly localised accumulation of other phytoalexins and of S0 in T. cacao and tomato xylem, suggest S0 is produced in sufficient amounts, at the right time and place in some interactions to contribute to resistance.