The heterogeneous form of nitrate reductase released from the membrane fraction of Escherichia coli by heat treatment was converted to a new electrophoretic form by incubation with trypsin. As a result of the trypsin treatment, the heat-released enzyme was converted from an associating-dissociating system to a nonassociating monomer (Mr approximately 200,000) which retained full enzymatic activity. Several distinct subunits in the 47,000- to 59,000-dalton range were converted to a single 43,000-dalton subunit during the trypsin treatment, while the other major subunit (155,000 daltons) was unaffected. Nitrate reductase extracted from the membrane fraction with deoxycholate and ammonium sulfate was composed of two apparently homogeneous subunits (155,000 and 59,000 daltons). The detergent-extracted enzyme preparation was converted by trypsin to an electrophoretic form very similar to the product of trypsin treatment of the heat-released enzyme with an identical subunit composition (155,000 and 43,000 daltons). These results demonstrate that the heterogeneous subunits present in the heat-released enzyme are produced during heat treatment by proteolytic cleavage of a single 59,000-dalton subunit. The fragments removed by trypsin treatment are implicated in the self-associating properties of the heat-released enzyme.