An x-ray fluorescence method for in vitro analysis of trace elements in biological samples is presented. The method is characterised by the use of an x-ray tube with secondary targets as a monoenergetic radiation source, and by 'infinitely thin' specimens. In the experimental work, different aspects have been examined in order to optimise the sensitivity of the method. It is shown that it is extremely important to use collimators of high purity materials and very pure and thin sample supports. Regarding the geometry, it is pointed out that a collimator between specimen and detector reduces the counting rate caused by scattering in air and other materials. Scattering in the biological matrix is reduced by preconcentration of biological liquids or tissues. The method has been applied to the analysis of blood serum samples. Typical sensitivities for Fe and Rb are 1.6 ng cm-2 and 1.5 ng cm-2 respectively, in a counting time of 10(3) s.