Preparations of broken Aspergillus nidulans hyphae contained both free and wall-bound autolysins. The bound enzymes were not solubilized by 8 M LiCl or neutral or anionic detergents; they were readily detached from walls by a cationic detergent or by autodigestion. Once detached, the enzymes did not reassociate with wall to give salt-resistant complexes. Six enzymes hydrolyzing wall polymers were bound to the envelope, and the same activities were also detected among soluble proteins in the cytoplasmic fraction. It is suggested that cytoplasmic vesicles, containing autolysins, are inserted into or trapped by newly formed wall in the growing hypha; these constitute the wall-bound autolysin fraction. Starvation for a carbon source derepressed the synthesis of five out of the six autolysins, and the amounts of both soluble and wall-bound activities increased by one to two orders of magnitude.