Four new isolates, identified asAlternaria tenuis, 34–016, 34–039, 35–056 and 35–193 were grown on revised tobacco medium. Mycelial and broth extracts were prepared and their biochemical and immunological properties were examined. Compared to the mycelial extracts, the four broth extracts had lower protein contents and showed fewer protein bands in isoelectric focusing, crossed-immunoelectrophoresis and sodium dodecylsulphate polyacrylamide gel electrophoresis. However, direct radioallergosorbent and passive cutaneous anaphylaxis tests with mouse IgE revealed that mycelial and broth extracts were of similar potency and had extensive cross-reactivity. In crossed-radioimmunoelectrophoresis using human atopic sera, the mycelial extracts showed 1–2 dominant and 3–4 minor allergens while the broth extracts had one dominant and one minor allergen. In RAST inhibition tests, two isolates, 34–039 and 35–056, had 100-fold more allergen content in the broth extracts compared to the mycelial extracts, whereas the levels were similar for broth and mycelial extracts of the other two isolates. The results indicated that culture broths of 34–039 and 35–056 were excellent starting materials for purification of some of the major allergenic components ofA. tenuisas they combined good yield with high allergenic activity, and lower level of other protein components. In addition, extracts containing wide spectrum of allergens and suitable for diagnostic purposes, can be prepared from mycelia and culture fluid of these isolates.