Tobacco (Nicotiana tabacum L.) microspores at the time of mitosis are characterized by the abundant occurrence of 92- and 98-kDa glycoproteins (GP92 and GP98). GP92 is a soluble protein while GP98 is bound to the insoluble microspore fraction. Both glycoproteins were isolated by affinity chromatography and SDS–PAGE and analysed by MS. Peptide sequences were determined by μ-HPLC/nano-ESI–MS/MS (electrospray ionization tandem MS). GP92 displayed homology to β-galactosidase (EC 3.2.1.23) and GP98 to β-xylosidase (EC 3.2.1.37) from Arabidopsis thaliana (L.) Heynh. The activities of the two enzymes in microspore and pollen extracts of tobacco exhibited similar developmental changes to the occurrence of GP92 and GP98, with a maximum around microspore mitosis. These two glycoproteins are the first identified enzymes characteristic of mitotic microspores. Arabidopsis transcriptomic data for five β-galactosidase and three β-xylosidase genes abundantly expressed in pollen were verified by reverse transcription–PCR of RNA from different stages of Arabidopsis pollen development and from various parts of the sporophyte. The results showed abundant expression of two genes (At5g20710, At1g31740) homologous to tobacco GP92 in microspores and early pollen, and of three genes (At5g56870, At2g16730 and At4g35010) in maturing pollen. Analysis of β-xylosidases showed abundant expression of a late pollen-specific gene At3g62710 and low expression of an early gene At5g10560. It is suggested that the early β-galactosidase and β-xylosidase genes may participate in cell wall loosening associated with pollen expansion after microspore mitosis and that the products of the late genes may play a role in cell expansion during pollen germination.