Development of the Drosophila embryonic midgut is dependent on a number of genes, including tinman and bagpipe, which are required for formation of the visceral mesoderm, and the homeotic genes and their targets, which act locally in the visceral mesoderm to direct formation of specific midgut constrictions. Here we report the identification and characterization of another gene, odd-paired (opa), required for normal midgut development. opa, first identified by its pair-rule mutant phenotype in the larval cuticle, encodes a protein containing putative DNA binding domains and other hallmarks of transcriptional regulators. We have positively identified the cloned gene as opa by mapping two null mutations to the open reading frame of the putative opa transcript; these mutations disrupt the open reading frame and generate predicted proteins lacking the DNA binding domain. We demonstrate that opa function is required for formation of the three characteristic midgut constrictions. To understand the mechanisms by which opa contributes to constriction formation, we have analyzed the expression and function of opa throughout midgut development. In the cellular blastoderm, opa is expressed ubiquitously in the ectoderm and mesoderm precursors throughout the presumptive segmented region. As development proceeds, opa expression ceases briefly both in the ectoderm and in the underlying mesodermal cells that later become the visceral mesoderm. We show that in opa mutants the visceral mesoderm is interrupted, evidently due to abnormal expression of bagpipe, a homeodomain gene required for the formation of the visceral mesoderm. At early stages of development in opa mutants, interruptions in the visceral mesoderm are observed at many positions along the anterior-posterior axis. As development proceeds, interruptions are less frequently observed; however, one interruption, coincident with the Antennapedia expression and variability of Ultrabithorax expression in opa mutants. From these observations, we infer that the loss of at least the first and second midgut constrictions in opa mutants is the result of defects first evident in the early stages of visceral mesoderm development.(ABSTRACT TRUNCATED AT 400 WORDS)