Factors, which affected the stability of peroxisomal membrane in vitro, were investigated. The decrease in the activity of catalase at neutral and alkaline pH was prevented by addition of Ca2+, Mg2+ and Ba2+; at the same time EDTA, EGTA and o-phenantroline decreased the stability of peroxisomes. All the effects were temperature-dependent. Ca2+ did not prevent the injury of peroxisomes during hypotonic lysis, but increased the electrostatic interactions of enzymes with the peroxisomal membrane. These data suggest that at neutral and alkaline pH the peroxisomal membranes had a large negative charge on their surface, which affected the stability of peroxisomes in vitro.