The aim of this work was to investigate the effects of total naphthenic acids and fractions of these complex mixture of acids obtained by fractionation based on acid ionization constants on rooting of in vitro grown chrysanthemum (Dendranthema x grandiflora Ramat. cv. Escort) shoots. Three concentrations (10, 50 and 100 μM) of the sodium salt of total naphthenic acids, its fractions gained by extraction on different pH (pH 2, pH 4, pH 7 and pH 9) and indole-3-butyric acid were examined. Thirteen of nineteen treatments gave significant increase of number of roots per explant compared to control. Six of fourteen treatments with naphthenic acids did not significantly differ from the best treatment with indole-3-butyric acid. The effect of all treatments with the highest concentration of naphthenic acids (100 μM) did not differ significantly from the effect of control. The rooting ability was evenly distributed among obtained fractions of naphthenic acids.