The Neurospora crassa cot-1 gene encodes a Ser/Thr protein kinase, which is involved in hyphal elongation. Many vacuoles, abnormally shaped mitochondria, and nuclei, along with differences in the structure of the cell wall and hyphal septa, were observed in hyphae of the cot-1 mutant shortly after a shift to the restrictive temperature. Immunolocalization experiments indicated that COT1 was associated with the cytoplasmic membrane; COT1 was also detected in the cytoplasm. The membrane-associated COT1 was absent from the cot-1 mutant when shifted to the restrictive temperature, as was a lower molecular weight isoform of COT1. We propose that COT1 may be involved in several cellular processes, and the spatial and temporal regulation of COT1 activity involves trafficking of the kinase within the fungal cell and its possible interaction with additional proteins.