A patient (KL) with progressive motor neuron disease associated with partial Hex A (alpha beta) and no Hex B (beta beta) activity, synthesized beta-chains which only associated with alpha-chains. To identify the molecular basis of this inability of beta-chains to self associate, RNA from cultured fibroblasts was reverse transcribed, the cDNA encoding the beta-chain amplified by polymerase chain reaction, subcloned, and sequenced to reveal two types of single missense mutation. The first mutation, (Type I) 619A----G, was paternally inherited and converted a 207IIe----Val in a highly conserved region believed to be associated with catalytic activity and activator protein binding. Biochemical evidence for impaired activator protein binding was obtained by purifying Hex A from KL urine and demonstrating a greater than 50% reduction of in vitro GM2 hydrolysis compared to normal urinary Hex A. In other cDNA species (Type II), a maternally inherited 1367A----C mutation converted 456Tyr----Ser in another highly conserved region of the beta-chain and we propose that this mutation leads to the inability of the beta-chains to self associate and thus reach maturity. These same cDNA species contained a second 362A----G mutation which converted 121Lys----Arg, but is apparently a polymorphism since it also occurs in some normal subjects. We propose that the patient is a compound heterozygote in which a combination of no self-association of the mutant beta-chains and impaired activator protein binding to alpha-beta (mutant) (Hex A) required for GM2 hydrolysis result in total beta-Hex B deficiency and slow accumulation of GM2 ganglioside, primarily in motor neurons.