In the course of our investigation into bioactive products of Alternaria porri (Ellis) Ciferri, the causal fungus of black spot disease in stone-leek and onion, we isolated tentoxin when the fungus was cultured in a stone-leek decoction for thirty days. Tentoxin is known as a non-specific toxin produced by some Alternaria species, i.e., A. tenuis, A. mali, A. citri, A. alternata, and A. longipes. From the chemotaxonomical point of view, it was of interest to us to study whether tentoxin is a characteristic metabolite in other Alternaria species than those already mentioned. We have already reported an HPLC determination method for tentoxin in an onion decoction of A. porri. This paper deals with the HPLC determination of tentoxin from a culture liquid of A. porri, A. solani, A. cucumerina, A. bataticola, and A. japonica, the causal fungi of plant disease in stone-leek, potato, cucumber, sweet-potato and radish, respectively. These were grown as artificial cultures (Richards and Czapek Dox with ZnSO₄) and natural culture with potato sucrose medium. The relationship between the culture medium and the productivity of tentoxin was also studied. The quantitative procedures were almost the same as those reported previously, using the internal standard method with α-naphthol as IS. Authentic tentoxin was dissolved in methanol with IS and subjected to HPLC. The concentration of tentoxin was calculated from the peak area using the previously reported work curve. Additional support for the identification of tentoxin was provided by LC/API-MS analysis in the case of A. bataticola, which showed a molecular ion peak of m/z 415 (M+H)⁺, confirming the molecular weight of tentoxin. The results obtained showed that tentoxin was detected in many of the Alternaria species tested, but its quantity depended upon the medium used. When Czapek Dox (with ZnSO₄) was used, tentoxin was produced in a high yield in A. porri and in a small amount in A. solani, but not detected in other tested Alternaria species. Hence, Czapek Dox was found to be most effective for producing tentoxin in A. porri. When Richards medium was used, considerable amounts (though varying) were detected in all tested Alternaria species except A. brassicae. When potato sucrose medium was used, tentoxin was not detected in A. japonica and A. brassicae, although small amounts were detected in other tested Alternaria species. Based on all these results, four Alternaria species (A. solani, A. cucumerina, A. bataticola, A. japonica) producing tentoxin can be newly added to the existing six species, and tentoxin is a non-specific toxin produced by common Alternaria species.