Some kinetic parameters of the human red cell Ca(2+)-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37 degrees C. After 30 min treatment with EGTA (1 mM) plus dithioerythritol (1 mM), a Vmax of about 0.4 mumol Pi/mg x hr and a Ks of 0.3 microM Ca2+ were found. When Mg2+ (10 mM) or Ca2+ (10 microM) were also added during preincubation, Vmax, but not Ks was altered. Ca2+ was more effective than Mg2+, thus increasing Vmax to about 1.3 mumol P/mg x hr. The presence of both Ca2+ and Mg2+ during pretreatment decreased Ks to 0.15 microM, while having no apparent effect on Vmax. Conversely, addition of ATP (2 mM) with either Ca2+ or Ca2+ plus Mg2+ increased Vmax without affecting Ks. Preincubation with Ca2+ for periods longer than 30 min further increased Vmax and reduced Ks to levels as low as found with calmodulin treatment. The Ca2+ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mM; leupeptin, 200 microM; pepstatin A, 100 microM; phenylmethanesulfonyl fluoride, 100 microM). The electrophoretic pattern of membranes preincubated with or without Mg2+, Ca2+ or Ca2+ plus Mg2+ did not differ significantly from each other. Moreover, immunodetection of Ca(2+)-ATPase by means of polyclonal antibodies revealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 microM), washing with EGTA (5 mM) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium.(ABSTRACT TRUNCATED AT 250 WORDS)