The influence of horseradish peroxidase (HRP) charge on Fab-HRP conjugates was investigated. Rabbit nonimmune Fab coupled via periodate or glutaraldehyde to Sigma HRP type VI (pI greater than 10) were cationic (positively charged) as determined by analytical isoelectric focusing. These conjugates and HRP type VI alone stippled the basal laminae and collagen fibers in Bruch's membrane of the rat eye in a pattern identical to anionic (negative) sites. Binding was not present after the anionic sites were removed by enzyme digestion prior to immunolabeling or when HRP type VIII (anionic with pI 3.6) was used in an Fab-HRP conjugate or in an unbound form. These results indicate that anionic HRPs should be used in Fab-HRP preparations if a nonspecific binding to anionic sites is possible.