In order to obtain reliable values of vitamin B_6 contents in some foods and food materials by microbioassay, it is critical to hydrolyze the samples satisfactorily prior to the determination .The conditions for the acid hydrolysis of samples were investigated with some plant and animal foods. It was found that pure pyridoxine-β-glucoside (PN-β-Glu), which was found in some plant foods, was hydrolyzed at 121℃ for 2 h with 0.88 N-HCl. Food samples from plant origins, to release pyridoxine (PN) completely from some plant matrix, must be hydrolyzed stronger conditions than the reported one, that a sample was hydrolyzed at 121℃ for 2 h with 0.44 N-HCl. It was also found that hydrolysis by HCl was rather suitable for the plant foods than by sulfuric acid. To save the time, three hour autocleave at 121℃ with 0.88 N-HCl for plant foods, and with 0.11 N-HCl for animal foods, was newly proposed as the conditions for the hydrolysis of foods satisfactorily.