Genetic research of fishes is hampered by lack of standardized karyotypes and reliable techniques of chromosome banding. The goal of this study was to develop a standardized karyotype of channel catfishIctalurus punctatusby a variety of banding techniques and computer-assisted analysis. Metaphase chromosomes were prepared from cultured leukocytes and kidney cells of adult and juvenile fish. Silver staining and alkali treatment methods were used to reveal nucleolus organizer regions (NOR) and the location of constitutive heterochromatin (C-bands). Chromosomes were treated with 10 different restriction enzymes, stained with Giemsa, and examined for banding patterns. The chromosomal data were analyzed with two computer software packages. The 29 chromosomes were classified into eight distinct groups based on morphology and size. The NOR were located on a pair of medium-sized submetacentric chromosomes (designated as D-11); this was consistent among cells from different specimens. The C-bands were small and restricted to centromeric regions and were useful for homologous pairing. A standard karyotype of CBG-banded chromosomes (C-bands visualized by treating with barium hydroxide and staining with Giemsa) was developed. The restriction enzymeMscI produced informative banding patterns with dark telomeric bands and clear centromeric regions. The enzymeHind III was most informative, yielding linear banding patterns that were consistent between members of homologous pairs. Our study provides a fundamental step in genome mapping of fishes. Standardization of the channel catfish karyotype and chromosome banding will facilitate physical mapping of genes in this important culture species.