Summary The action of the enzymes novozym 234, chitinase and zymolyase 20T on the yeast‐like cells of Paracoccidioides brasiliensis was studied in an attempt to obtain protoplast release. Three enzyme systems were used: the first consisted of novozym 234 and chitinase plus 0.2 m phosphate buffer, 0.9 m sorbitol and 0.5 m sodium thioglycolate; the second consisted of novozym 234, chitinase, zymolyase 20T, buffer and osmotic stabilizer, with no sodium thioglycolate; the third consisted of the same enzymes as used in the second system but at twice the concentration, plus buffer and osmotic stabilizer. Protoplasts were only released from 72‐hold cells cultured on solid peptone‐yeast extract‐glucose medium (PYG) treated with the third enzyme system. Sodium thioglycolate used as pretreatment favoured protoplast release but had no such action when added to the enzyme solution, possibly by altering the activity of the enzymes, novozym 234 in particular. The osmotic stabilizer used, 0.9 m sorbitol, was probably one of the factors, in addition to the enzymes, responsible for the cytoplasmic changes observed by transmission electron microscopy in yeast phase cells and in their protoplasts.