Alternative splicing is an important mechanism contributing to the increased proteome diversity in higher eukaryotes. We have explored the alternative splicing events in the Drosophila longitudinals lacking (lola) gene by means of 5' RACE, 3' RACE, genome sequence searches, and EST sequencing. We demonstrated that the lola locus is comprised of 32 exons spanning over 60 kb, and encodes a total of 80 alternatively spliced variants consisting of 5' and 3' variable sequences and constitutive common exons. All the variants shared a common sequence (exons 5-8) encoding the N-terminal region containing the BTB domain, but both the 5' and 3' ends were variable. There were four promoters responsible for the variation in the 5' end (exons 1-4). Alternative splicing was involved in the variation in the 3' end corresponding to the C-terminal variable region, which was encoded by one or two exons that were selected from 20 groups of exons in a mutually exclusive manner (exons 9-32). Seventeen of the 20 isoforms contained C(2)H(2)-like zinc finger motifs in the C-terminal variable region. Analyses of the 3' variant-specific cDNA pools revealed that all combinations of 5' and 3' variable sequences were expressed in both the embryonic and third instar larval stages. Since the BTB domain mediates dimerization, lola encodes a family of transcription regulators with a large variety of DNA- or protein-binding specificities, and could be involved in various developmental processes, including the embryonic neural pathfindings. We also showed that the structures of Lola isoforms were highly conserved in Drosophila pseudoobscura.