We have shown previously that transcription of the Drosophila homeotic gene Antennapedia results in four major RNA species which differ in long 5'- and 3'-untranslated sequences. The protein-coding portion of these transcripts, however, is located in exons common to all. Using RNase protection assays and further cDNA clone isolation, we have now detected two alternative splicing events between exons of this region. These result in four RNA variations which, if translated, would encode a family of Antennapedia proteins. By analyzing transcripts from various developmental stages and isolated tissues, we show that alternative splicing is under strict temporal and spatial regulation. For example, while similar patterns of splicing were found for all wild-type thoracic imaginal disks examined, these differed distinctly from the patterns observed in neural tissues. Our results suggest that individual RNAs may be associated with different biological roles, and provide molecular evidence that the Antennapedia gene is involved in multiple functions.