The aim of the investigation was to test the reproducibility and accuracy of a new method to measure temporomandibular joint (TMJ) fluid concentrations of various substances by saline washing, using exogenous B12 as a marker. An in vitro test was first performed with glucose as a test substance. The difference between a B12-calculated and known standard concentration of glucose was very small. Saline washing of the TMJ was performed on 13 patients having signs of TMJ arthritis, and the aspirates obtained were analyzed for neuropeptide Y-like immunoreactivity (NPY-LI) and interleukin-1 beta (IL-1 beta). Vitamin B12 was mixed with the saline immediately before injection, and a sample of the aspirate was later compared photometrically with the injection solution. There were positive correlations between saline aspirate and joint fluid concentrations for NPY-LI and IL-1 beta, and the correlations were stronger for saline aspirates with high joint fluid content. This study shows that the method is reliable for measurement of joint fluid concentrations of various substances, such as NPY-LI and IL-1 beta.