The author has investigated the influences of a so-called new vitamin B group comprising orotic acid, thioctic acid, bantothenic acid, adenine, and carnitine at the optimal medium concentrations on the number of megakaryocytes and their functions of motility and platelet separation of normal rat bone marrow cultured in vitro, and the results obtained are as follows. 1. The number of megakaryocytes appearing in the outgrowth was increased with adenine, carnitine, pantothenic acid, orotic acid, and thioctic acid in this declining order. 2. Acceleration of the functions of megakaryocytes was observed with carnitine, adenine, pantothenic acid, thioctic acid, and orotic acid, in this declining order.