A primary cell line (designated as CCf) derived from caudal fin tissue of channel catfish, Ictalurus punctatus, was developed using explant techniques. The cell line grew fastest in media supplied with FBS and channel catfish serum. The duplication time of the cell line under optimal conditions was ∼56 h at a plating density of 1.1 × 10(5) cells/ml. The cell line has been propagated continuously for 25 passages (1:4 dilution per passage), cryopreserved, and recovered successfully at different passages. The cultured cells had fibroblastic morphology, and synthesized fibronectin and Type I and III collagens in the cytoplasm. The cell line maintained the normal diploid chromosome number (58) of channel catfish throughout the experiment. Nucleolus organizer regions were located on the short arms of a pair of medium-sized submetacentrics, which is typical for channel catfish. This study provides a method for acquiring a cell line from juvenile catfish without sacrifice, and is especially useful for early screening of valuable fishes.