Abstract Aspergillus awamori and certain other Aspergillus and Penicillium species accumulate the α-glucan, nigeran, in their hyphal walls when shifted to a growth medium deficient in nitrogen. A. awamori hyphae, actively synthesizing negeran, were converted to protoplasts by digestion with a lytic enzyme mixture and the regeneration process was observed. Germ tubes were evident within 3 to 5 h and by 9 h hyphal development was extensive. Freshly prepared protoplasts were immediately active in biosynthesis of new wall and capable of transporting and utilizing exogenous substrates as evidenced by their loss of lytic sensitivity within 1 to 3 h of reversion and by the fact that incubation of new protoplasts with [ 3H]glucose resulted in incorporation into cellular products within 10 min. Among these products was a (β-1,3)-glucan fraction. Accumulation of nigeran, also monitored by incorporation of radioactivity from [ 3H]glucose, did not occur until 12 to 24 h into the regeneration period and was correlated with the observed reversion of the protoplasts to a hyphal mode of growth. Thus, although protoplasts were prepared from hyphae synthesizing nigeran, they did not continue to do so immediately at the start of regeneration. Synthesis of other wall polymers and/or attainment of a particular cell shape precede nigeran deposition in the regenerating protoplasts, a result consistent with a probable role for nigeran as a secondary wall polymer. A monoclonal immunoglobulin A conjugated to phenylisothiocyanate and specific for dextrans with (α-1,3) linkages has been utilized to detect glucans of this type on the surface of the regenerating protoplasts.