Sera from obligate heterozygotes for deficiency of the C8β subunit of the eighth component of human complement (C8) were analyzed for the molecular composition of C8. The C8α-γ and C8β subunits were separated by SDS-PAGE, visualized by immunoblotting, and the resulting bands were quantitated by laser densitom-etry. The laser densitometric absorption data were set to 100 arbitrary units (AU) for both subunits of pooled normal human sera. The AU values of individual normal sera ranged from 45 to 150 AU for C8α-γ (median 99 AU) and from 45 to 140 AU for C8β (median 101), whereas the C8α-γ/C8β-ratio varied from 0.7 to 1.4. Sera from C8β-deficient heterozygotes differed, as expected, from the normal sera for the markedly reduced levels of C8β (20 to 90 AU, median 55 AU) and for the higher C8α-γ/C8β-ratio (1.3 to 3.5). High voltage agarose gel electrophoresis was used to separate free and C8β-bound C8α-γ. The migration of free and C8β-bound C8α-γ subunit was checked by hemolytic overlay gels and by second dimension SDS-PAGE and immunoblotting. Immunochemical evaluation of C8α-γ using this system revealed about 5–14% free C8α-γ in sera with normal C8 and higher levels, from 33–71%, in the C8βD heterozygous sera. Functional analysis confirmed the substantial increase of free C8α-β in the heterozygous group. We conclude that the C8 in C8βD heterozygous sera is characterized by increased amounts of free C8α-γ due to reduced concentrations of the C8β subunit. This finding may help to identify individuals heterozygous for C8β deficiency.