A cDNA library of Brassica napus (cv. Westar) was constructed using poly(A)+ RNA isolated from developing anthers of flower buds 2-3 mm in length. Differential hybridization, using cDNA probes complementary to poly(A)+ RNA from developing anthers or seedlings, was used for initial screening. In addition to Southern and Northern blot analyses of selected clones, RNA-PCR assays and in situ hybridization were used to study the temporal and spatial gene regulation in anthers at the transcriptional level. Five independent cDNA clones, showing no cross-hybridization to one another, were characterized, and their expression patterns could be grouped into three distinct categories. Two cDNA clones, BA112 and BA158, are tapetum-specific: the corresponding mRNAs accumulate in young anthers and decline as the tapetum cells degenerate later in anther development. The transcripts represented by BA54 and BA73 accumulate late in anther development and reach a maximum level in mature anthers prior to anthesis; BA54 has been confirmed to be pollen-specific. The third category, represented by BA42, is found to encode a protein sharing 64-67% amino acid similarity with chalcone synthase (CHS) from various plant species; the transcript is localized in the peripheral cells of the vascular bundle, tapetum, and developing microspores.