Technical Abstract: Calcein marking and cohabitation challenge have not been investigated in fish parasite research. This study evaluated a cohabitation challenge method in immunization trials against Ichthyophthirius multifiliis (Ich) using calcein, a fluorescent dye, to mark channel catfish Ictalurus punctatus (Rafinesque). Fish were marked by calcein immersion at 0, 500 and 1500 mg L-1, respectively, and then challenged with 10,000 theronts per fish. No difference was noted on fish infection level, mortality and mean day to death (MDD) caused by Ich between fish marked and non-marked with calcein or between marked with high (1500 mg L-1) and low (500 mg L-1) concentration of calcein. After ensuring that calcein marking had no effect on the susceptibility of fish to Ich theronts, two immunization trials were conducted to evaluate the cohabitation challenge model using calcein-marked catfish. Fish mortality, relative percent survival (RPS), and MDD were compared between cohabitation-challenged fish and fish challenged by non-cohabitation. No significant difference was observed in RPS for cohabitation-challenged fish and fish challenged by non-cohabitation. Cohabitation challenge can be used as an alternative challenge method for parasite study since it closely mimics natural exposure.