Background. Prodigiosin is a red pigment produced as a secondary metabolite by Serratia marcescens , characterized with unique tripyrrole structure and exhibits such pharmacological characteristics as antitumor, antimicrobial, antioxidant, immunodepressant, and also used in various branches of biotechnology as a natural dye or marker of petroleum products. However, pigmentation is only present in a small percentage of isolated cultures among different S. marcescens strain, therefore, the effectiveness of biotechnological productivity is primarily determined by the producer strain, as well as the rational conditions for biosynthesis. Pigment production levels are strongly influenced by various factors, such as the set and ratio of the components of the culture medium (concentration of a particular substrate, C:N ratio) and cultivation conditions (temperature, pH). Besides, the use of relatively low-cost substrates is an important condition for reducing financial costs for the production of culture media and increasing the level of biosynthesis of the target product. Therefore, the determination of the influence of the culture medium for the strains of S. marcescens and the identification of the main factors contributing to the accumulation of the pigment prodigiosin to develop biotechnologies for the production of drugs for therapeutic purposes is relevant. Objective. The purpose of the paper is investigation of pigment-forming strain of S. marcescens under cultivation conditions on the surface of solid nutrient media, determination of biochemical features, and rational cultivation conditions that contribute to the accumulation of the pigment prodigiosin. Methods. The strain of S. marcescens isolated from the bentonite clays of Kurtsivskyi deposit (Crimea, Ukraine) was cultured in Petri dishes on solid nutrient media with different temperatures, pH, carbon and nitrogen sources. The influence of various values of the studied factors was established by the level of pigment accumulation, which was determined by the gravimetric method. The acidity of the nutrient medium was determined by the potentiometric method. Results. The influence of the initial pH values of solid nutrient medium, temperature, and various sources of carbon and nitrogen on the accumulation of pigment during the cultivation of the strain S. marcescens was studied. The temperature, pH, and compounds, sources of carbon and nitrogen, favorable for obtaining the pigment, were determined. Conclusions. It was found that the rational parameters for cultivation are the temperature +27–28 °C and the acidity of the nutrient medium of 6–7, and the best sources of carbon and nitrogen for prodigiosin accumulation are glycerin and peptone (as well as casein hydrolysate, yeast extract or ammonium (nitrate) inorganic compounds), respectively.