Abstract A total-reflection X-ray fluorescence (TXRF) spectrometer has been designed for an 18 kW rotating anode system. The spectrometer uses a multilayer mirror, which is especially advantageous in the analysis of biological samples, and a charge-coupled device (CCD), which is an aid in the alignment of the instrument. To ensure spectrometer stability, the collimator, multilayer mirror and sample holder are connected directly to the housing of the rotating anode while the detector rests on a separate support. With the CCD, the intensity distribution of the X-ray beam was measured; and the beam thickness at the sample position was calculated. A relative sensitivity calibration curve was established for the spectrometer and shown to agree well with a theoretical expression. The calibration was validated by measurements on a certified reference material of plankton. The measurements also provided information about the accuracy, precision and detection limits of the spectrometer for biological samples.