In order to identify the individual contributions of the kringle (K) domains of human plasminogen (Pg) to the epsilon-aminocaproic acid (EACA) induced stimulation of Pg activation by low-molecular-weight urokinase-type plasminogen activator (LMW-uPA) and inhibition of this same activation by Cl-, we constructed the most conservative recombinant- (r-) Pg mutants possible that would greatly reduce the strength of the EACA binding site in the omega-amino acid binding kringles, [K1Pg] ([D139-->N]r-Pg), [K4Pg] ([D413-->N]r-Pg), and [K5Pg] ([D515--N]r-Pg). In each case, this involved mutation of a critical Asp (to Asn) within these three kringle domains in intact Pg. The three r-mutants were expressed in r-baculovirus-infected lepidopteran insect (Trichoplusia ni) cells. In the presence of Cl-, the positive activation effector, EACA, first stimulated and then inhibited the LMW-uPA-catalyzed initial activation of wild-type (wt) r-[Glu1]Pg and, to a lesser extent, the [K5Pg] mutant, [D518-->N/Glu1]r-Pg. The concentration of EACA that produced 50% stimulation of activation (C50) occurred at 3.3 mM for wtr-[Glu1]Pg and at 0.7 mM for [D518-->N/Glu1]r-Pg. Subsequent inhibition by EACA occurred with a C50 of approximately 15 mM and is likely due to inhibition of the amidolytic activity of plasmin generated during the activation. Similar initial activation rates of both [D139-->N]r-Pg and [D413N]r-Pg did not display this initial EACA-mediated stimulatory phase but did undergo ultimate inhibition with a C50 for this process that was similar to wtr-[Glu1]Pg and [D518-->N/Glu1]r-Pg.(ABSTRACT TRUNCATED AT 250 WORDS)