Biotinylation of cell surface proteins is often used as an alternative to radioactive labelling procedures, but very little is known about the labelling of the different allelic products of polymorphic antigenic systems. In this report, we demonstrate that NHS-LC-biotin labels bovine MHC class II molecules with different efficiencies for several allelic forms of this polymorphic system compared to conventional metabolic labelling with [35S]methionine. This was shown after immunoprecipitation and one-dimensional isoelectric focusing (1D-IEF). The avidity of the monoclonal antibody Bo139 (alpha-bovine MHC class II) was not affected after in situ biotinylated of bovine PBMC, as revealed by flow cytometric analyses, immunoblotting after SDS-PAGE and immunoprecipitation. The biotinylation did not affect the apparent isoelectric points of polymorphic bovine MHC class II beta chains. This was demonstrated by double labelling of cells with [35S]methionine and subsequent biotinylation and comparison of the banding pattern after immunoprecipitation and 1D-IEF. 1D-IEF of 11 unrelated animals resulted in the demonstration of 29 polymorphic bands of which eight were detected by both labelling procedures, six only after biotinylation and 15 only after metabolic labelling with [35S]methionine. Hence, biotinylation alone cannot serve as an alternative for radioactive labelling of bovine MHC class II molecules but can reveal expressed allelic forms not detectable by metabolic labelling with [35S]methionine.