A favorable condition was reexamined for the determination of thiamine by cup-plate method using Lactobacillus fermenti. The size and sharpness of periphery of zone were influenced by various biological and physicochemical factors, such as pHs of medium and thiamine solution or times of diffusion of test solution into the plate. Therefore, the procedure must be done under a definite condition. The following condition was preferred by the authors. Assay medium : Sarett・Cheldelin's with 1% agar, 1.5mm plate thickness, pH 5.7〜6.1. Thiamine solution : pH 5.6〜6.0. Incubation : 37℃, 16〜18hrs.