Abstract Challenge methods for inducing nocardiosis, caused by Nocardia seriolae in yellowtail, Seriola quinqueradiata , were evaluated. The first method involved intraperitoneal injection of 1.1 × 10 2 , × 10 3 and × 10 4 cfu of N. seriolae ; the second was by bath immersion with three different concentrations of bacterial suspension for 10 min; the third was by intradermal injection using a multipuncture device; the fourth was by oral administration using a tube; the fifth was based on the co‐habitation of uninfected fish with others that had been artificially infected, i.e. intraperitoneally or by immersion for 10 min. The LD 50 values for the infection trials were 1.9 × 10 2 (intraperitoneal injection), 1.5 × 10 4 mL −1 (immersion), 4.3 × 10 6 (intradermal injection) and 1.7 × 10 7 (oral administration). In the co‐habitation challenge, mortalities were 70% and 50% in groups of non‐infected fish mixed with fish infected by the i.p. injection and immersion methods, respectively. Fish challenged by intraperitoneal injection developed adhesions. Fish challenged by other methods did not show any gross clinical signs and moribund fish were similar to natural infection.