MASP-2 cleaves C4 and C3 in the complement lectin pathway, but how MASP-2 is activated is not yet known. We developed two assays for sensitive, specific measurement of MASP-2 in normal human serum (NHS), which were used to isolate MASP-2 from, and investigate its mechanism of action in, NHS. Here, we report that both C3 and α2M have major roles in MASP-2 and lectin pathway activation in NHS. MASP-2 isolated from either MBL-depleted or unmodified NHS by sequential DEAE and CM column chromatography eluted from Sephacryl-300 at ∼800 kD in saline-EDTA (>800-fold purification; ∼32% yield). The MASP-2 activity was resolved into two peaks on S-300 in high salt-EDTA (HS-EDTA), complexed with α2M in the heavier, and with C3 in the lighter, peak. Treatment of NHS with methylamine or hydrazine, or removal of C3 by passage through anti-C3 columns, resulted in loss of ability of mannan-MBL complexes to activate MASP-2 in NHS; this was reconstituted by purified C3. Readdition of α2M alone had no effect, but α2M enhanced reconstitution by C3 in methylamine-treated serum. Methylamine and hydrazine had no effect on preformed MBL-MASP-2 complexes. MASP-2 complexes with α2M and C3 were present in unmodified and methylamine-treated NHS in both saline and HS-EDTA, while MBL-MASP-2 complexes were present only with calcium in unmodified NHS. MBL purified on immobilized mannan columns from methylamine-treated NHS had no associated MASP-2 activity, unlike MBL purified from unmodified NHS. Methylamine also prevented MASP-2 activation in normal mouse serum, but MASP-2 activation was only partially reduced in C3- and α2M-knockout mice. We conclude that MASP-2 is bound with α2M and C3 as well as MBL in NHS, and suggest that α2M is a transport protein for MASP-2 and C3 acts as a cofactor in MASP-2 activation i by MBL-mannan complexes in normal human serum.