We recently reported that Serratia 56K protease is inhibited by plasma alpha 2 macroglobulin (alpha 2M) temporarily and by chicken egg white ovomacroglobulin (ovoM) continuously (Molla, A. et al. (1986) Infect. Immun. 53, 522-529). The inhibition of this protease is almost complete with ovoM whereas it is incomplete with alpha 2M, although these two macroglobulins show homology and many similarities. In the present study we determined the apparent numbers of binding sites and binding constants for the two macroglobulins by means of the fluorescence polarization method using FITC-labeled 56K protease. The time courses of complex formation of 56K protease with alpha 2M and ovoM were different; with ovoM it was complete within 5 min while with alpha 2M 150 min was required. Their apparent molecular volumes were also different; the fluorescence polarization value of the E/I complex was 18.7% larger with ovoM than with alpha 2M. The association constants obtained on Scatchard plot analysis with 56K protease and alpha 2M or ovoM were 0.33 X 10(7) M-1 and 1.09 X 10(7) M-1, respectively. One molecule of each of these macroglobulins binds 1.13 and 1.35 molecules of 56K protease, respectively. Upon E/I complex formation, an increase in amino groups due to proteolysis was noted in both cases, but more progressive proteolysis was observed in the case of alpha 2M. Furthermore, when the 56K protease was inactivated through the depletion of Zn atoms, complex formation did not occur.