Because of conflicting reports on plasma levels of total immunoreactive glucagon (IRG) in obesity, we investigated, by gel filtration, the individual IRG fractions in grossly obese patients (N = 16; BMI 40 ± 2) as compared with normal weight subjects (N = 18). Mean total IRG was 142 ± 12 pg/ml in normal subjects and 190 ± 15 pg/ml (P < 0.02) in obese subjects. IRG³⁵⁰⁰ was 16 ± 2 pg/ml in normal subjects and 34 ± 5 pg/ml (P < 0.005) in the obese subjects. Obese patients with normal glucose tolerance demonstrated 30 ± 5 pg/ml IRG³⁵⁰⁰ (N = 11; P < 0.01) as compared with those who were obese with impaired glucose tolerance (42 ± 12 pg/ml, N = 5; P < 0.0025), although the total IRG of the latter group (173 ± 31 pg/ml) was not different from that of normal subjects. IRG⁹⁰⁰⁰ was of comparable magnitude in the entire groups of obese and normal subjects. A wide variability of big plasma glucagon levels was found in all three groups, mean levels not being significantly different. These data suggest that plasma IRG³⁵⁰⁰ levels are elevated in grossly obese patients, particularly in those patients with impaired glucose tolerance, a finding that may be masked by mere measurements of total plasma IRG.