The concentrations of itraconazole and its active metabolite, hydroxy-itraconazole, were determined in the sera of 49 patients having received itraconazole for therapeutic or prophylactic purposes. Measurements by HPLC were compared with a biological assay using a strain of Paecilomyces variotii as test organism and itraconazole as standard. The concentration of hydroxy-itraconazole in serum was on average 1.6 times higher than the concentration of itraconazole. The sum of itraconazole and hydroxy-itraconazole concentrations determined by HPLC showed a linear relationship with the concentrations obtained by the bioassay (r = 0.94). The biological method gives approximately 20% higher values than the HPLC method indicating that hydroxy-itraconazole is biologically more active than itraconazole. As the values of the Paecilomyces variotii bioassay are in the same range as the HPLC measurements, it is concluded that both methods are suitable for the monitoring of itraconazole medication.