C3 conversion by trypsin was investigated with anti-C3a, anti-C3B, anti-C3c and anti-C3d sera in single radial immunodiffusion. In sequence, C3B determinants disappeared within 1 min of incubation and C3a vanished after 5 to 8 min. C3c and C3d, however, increased in concentration. The loose complex C3ab generated after the disappearance of C3B could be dissociated by lowering the pH and by subjection to electrophoresis. Free C3a production, demonstrated by means of rocket electrophoresis against anti-C3a, skin tests, and gel filtration with radioactively labeled C3, was maximal after 1 min of trypsin treatment. Isolated C3 activated by trypsin in solution was bound to erythrocytes in a C3b form as could be demonstrated by agglutination with anti-C3c and anti-C3d. This was also the case with C3 bound to erythrocytes after incubation of cold agglutinin serum with fresh normal serum at pH 7.5. Both of these C3 products proved inactive in the biologic test systems used. In contrast, isolated C3 activated by bound and fixed to the cell surface was biologically active. cells obtained in this way were agglutinated not only by anti-C3c and anti-C3d, but also by anti-C3B and anti-C3a sera. The C3 conformation which is assumed to be characterized by the C3B determinants seems essential for the biologic properties. Nevertheless, the C3B antigenicity is not directly related to the biologic activity of C3. Since C3a was detected on cells as well as on IgG1, its activity might possibly be evolved on the surface of an immune complex. From the antigenic properties of C3 during conversion and binding to both erythrocytes and immunoglobulins by trypsin and treatment, it is concluded that both enzyme activities attack the same region of native C3. The antigenic properties of C3 after binding depend on whether the converting enzyme is present in solution or in a solid phase.