Genome searches in this study indicate that the nematode Caenorhabditis elegans genome has 2582 bidirectionally oriented genes that account for more than 25% of the total genes. We analyze the transcriptional repression system for one of these predicted bidirectional promoters, which controls the expression of the spermathecal gene sth-1 and collagen gene col-43. These two genes are separated by 1.3 kb and are transcribed bidirectionally. sth-1 is expressed in spermatheca after the L4 stage and col-43 is expressed in the hypodermal cells of the L2d dauer stage. The upstream regions required for the expression of sth-1 and col-43 shared an overlapped control sequence. Two homeoproteins, MAB-18 and CEH-14, were isolated by yeast one-hybrid screening as binding proteins of the overlapped region. MAB-18 bound to two homeodomain-binding sites and interacted with CEH-14 to repress col-43 expression in spermatheca. These results indicate that the two homeoproteins interact with each other to repress col-43 expression in sth-1-expressing tissues. This is the first report of bidirectional gene regulation analysis in the C. elegans genome. (c) 2005 Elsevier Ltd. All rights reserved.