 Insertion sequences associated with " Mycobacterium ulcerans", IS2404 and IS2606, were detected by PCR.These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 °C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 μg ml–1), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide.On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, "Mycobacterium pseudoshottsii" sp.