Mycelia from four mutants of Aspergillus nidulans impaired in penicillin production at separate genetic loci were treated with an enzyme complex capable of lysine cell walls, then mixed in all possible paired combinations and grown in osmotically buffered penicillin production media, containing 2-deoxyglucose and an unrefined mixture of polyoxins to prevent cell wall regeneration. The culture filtrates were assayed after 6 d and significant penicillin yields were observed in four of the six possible combinations. None of these pairs produced penicillin when grown together as normal mycelium, suggesting that intermediates of the penicillin biosynthetic pathway unable to diffuse from untreated mycelium could do so from enzyme-treated mycelium when cell wall regeneration was inhibited. A general method is thus available for examining biochemical pathways with mutants accumulating intermediates unable to cross the cell wall barrier.