Properdin factor D (GBGase, C3Pase) is a 25,000 dalton protease required for alternative pathway activation. After isoelectric focussing of normal human serum (NHS) in polyacrylamide gel, an agar gel overlay containing 1.7% guinea pig erythrocytes and 10% NHS in 0.05 M barbital buffer with 1 mM mg++ and 5 mM EGTA developed lysis bands for C3, factor B and one other protein with a more basic isoelectric point. The latter (but not bands of C3 or B) is also produced in an overlay containing factor D-depleted serum instead of NHS (Martin et al.). In both these assays, purified human α, β/γ, and autocatalyzed thrombin (T) produced lysis bands corresponding to protein distribution. The major band of autocatalyzed, but no other T, coincided with factor D. Incubation of β/γT in NHS resulted in no noticeable loss of intensity of the lytic bands.