S100A12 is a calcium-binding protein predominantly found in neutrophil granulocytes and monocytes. Its usefulness in monitoring inflammatory disease states depends on documentation that assay results are reliable. This study aimed at defining guidelines for blood sampling, selection of optimal material handling and reference intervals in healthy controls while taking into account the basic features of S100A12. An enzyme linked immunosorbent assay was developed based upon antibodies induced in rabbits by injection of recombinant S100A12. Our studies confirm that oligomers of S100A12 are generated in the presence of calcium. Structural changes in S100A12 mediated by calcium influence the interaction with antibody. This is proposed as the background for our very low readings of S100A12 in Ethylene Diamine Tetraacetic Acid (EDTA) plasma. Individual S100A12 levels did not change substantially over a 5-week sampling period. Based upon testing of 150 blood donors we suggest reference intervals of S100A12 in serum to be 49–1340 μg/l for women and 27–1750 μg/l for men. The estimated mean concentrations were 234 μg/l in serum samples (range 12–15791), 114 μg/l (range 3–17282) in re-calcified EDTA plasma and 48 μg/l (range 2–14843) in heparin plasma. Without adding calcium to EDTA plasma before running the assay, concentrations were around 2 μg/l (16 persons). S100A12 quantification is assumed to become relevant for diagnostic use in many disease states. The importance of the handling and analysing conditions for a reliable result was examined. We recommend serum collected in gel-containing tubes as the preferred sample material and have suggested reference intervals for healthy individuals.