Immobilon-P Transfer Membrane (IM), a hydrophobic membrane, has been tested as a substrate for immunoblotting (Western blotting) in a routine serological assay for human immunodeficiency virus (HIV). Comparison of IM to nitrocellulose (NC) has shown the following advantages: (a) better retention of total HIV antigens, (b) improved detection of antibodies to glycosylated envelope antigens, and (c) ease of membrane handling. As a direct result of these improvements, less HIV antigen is required for detection of HIV antibodies in immunoblotting using the IM. Additionally, in some cases, HIV infection can be confirmed earlier due to enhanced detectability of the envelope antigens. Sera from 444 subjects including 4 seroconversions and 4 AIDS patients were investigated.