The filamentous fungus Neurospora crassa undergoes a well-defined developmental program, conidiation, that culminates in the production of numerous asexual spores, conidia. Several cloned genes, including con-10, are expressed during conidiation but not during mycelial growth. Using a previously described selection strategy, we isolated mutants that express con-10 during mycelial growth. Selection was based on expression of an integrated DNA fragment containing the con-10 promoter-regulatory region followed by the initial segment of the con-10 open reading frame fused in frame with the bacterial hygromycin B phosphotransferase structural gene (con10-hph). Resistance to hygromycin results from mutational alterations that allow mycelial expression of the con-10-hph gene fusion. A set of drug-resistant mutants were isolated; several of these had abnormal conidiation phenotypes and were trans-acting, i.e., they allowed mycelial expression of the endogenous con-10 gene. Four of these had alterations at a single locus, designated rco-1 (regulation of conidiation). Strains with the rco-1 mutant alleles were aconidial, female sterile, had reduced growth rates, and formed hyphae that coiled in a counterclockwise direction, opposite that of the wild type. The four rco-1 mutants had distinct conidiation morphologies, suggesting that conidiation was blocked at different stages. Wild-type rco-1 was cloned by a novel procedure employing heterokaryon-assisted transformation and ligation-mediated PCR. The predicted RCO1 polypeptide is a homolog of Tup1 of Saccharomyces cerevisiae, a multidomain protein that mediates transcriptional repression of genes concerned with a variety of processes. Like tup1 mutants, null mutants of rco-1 are viable and pleiotropic. A promoter element was identified that could be responsible for RCO1-mediated vegetative repression of con-10 and other conidiation genes.Neurospora crassa, a filamentous fungus, has three distinct sporulation pathways that lead to the production of either of two types of asexual spores, macroconidia and microconidia, or sexual spores, ascospores. Formation of macroconidia, known as macroconidiation or conidiation (as it will be referred to throughout this paper), has been extensively studied at the morphological level (32,40,53,59,64). Upon receipt of certain environmental cues, e.g., carbon starvation, vegetative tissue, the mycelium, initiates conidiation by producing aerial hyphae that grow away from the nutrient surface. These hyphae undergo apical budding, generating what have been termed minor constriction chains. Subsequent growth by continued apical budding leads to hyphal segments with more pronounced, major constrictions. These proconidial chains continue to differentiate by completing septation at the sites of major constrictions. Just prior to disarticulation of mature conidia, a fragile structure, termed the connective (59), is evident. The connective joins adjacent proconidia and is ultimately cleaved. The entire process of conidiation takes 12 to 24 h. Microc...