Neurospora crassa is a filamentous fungus that grows on semisolid media by forming spreading colonies. Mutations at several loci prevent this spreading growth. cot-1 is a temperature sensitive mutant of N.crassa that exhibits restricted colonial growth. At temperatures above 32 degrees C colonies are compact while at lower temperatures growth is indistinguishable from that of the wild type. Restricted colonial growth is due to a defect in hyphal tip elongation and a concomitant increase in hyphal branching. We have isolated a genomic cosmid clone containing the wild type allele of cot-1 by complementation. Sequence analyses suggested that cot-1 encodes a member of the cAMP-dependent protein kinase family. Strains in which we disrupted cot-1 are viable but display restricted colonial growth. Duplication, by ectopic integration of a promoter-containing fragment which includes the first one-third (209 codons) of the structural gene, unexpectedly resulted in restricted colonial growth. Our results suggest that an active COT1 kinase is required for one or more events essential for hyphal elongation.