This study was conducted to determine whether multivalent, precipitating antigens are required for formation of subepithelial electron-dense immune deposits in glomeruli. 2-nitro-4-azidophenyl (NAP) was conjugated with variable density to human serum albumin (HSA) to yield nonprecipitating (NAP3.1 X HSA and NAP11.4 X HSA) and precipitating (NAP19.7 X HSA) antigens with antibodies to the hapten. These antigen preparations were cationized with ethylene diamine to enhance deposition in renal glomeruli due to interaction with the fixed negative charges in the glomerular capillary wall. Following injection into the left renal artery of rats these antigens alone persisted in the glomeruli for a relatively short time by immunofluorescence microscopy. When antibodies to NAP were injected intravenously after the antigen injection, the nonprecipitating antigens and antibodies were detectable in the glomeruli by immunofluorescence microscopy up to 8 h, comparable to antigen alone. Electron-dense deposits were not formed in these glomeruli. In contrast, when the precipitating antigen was injected and followed by antibodies to the hapten, antigen and antibody were detected by immunofluorescence microscopy through 96 h. In these specimens electron-dense deposits were present from 40 min through 96 h and after 24 h the deposits were present only in the subepithelial area. The same results were obtained when the nonprecipitating hapten-carrier conjugates were followed with antibodies to the carrier molecule. These data indicate that the persistence of immune deposits by immunofluorescence microscopy and the formation of electron-dense deposits in the subepithelial area require a precipitating antigen-antibody system.