Reference values are used to describe the dispersion of variables in healthy individuals. They are usually reported as population-based reference intervals (RIs) comprising 95% of the healthy population. International recommendations state the preferred method as a priori nonparametric determination from at least 120 reference individuals, but acceptable alternative methods include transference or validation from previously established RIs. The most critical steps in the determination of reference values are the selection of reference individuals based on extensively documented inclusion and exclusion criteria and the use of quality-controlled analytical procedures. When only small numbers of values are available, RIs can be estimated by new methods, but reference limits thus obtained may be highly imprecise. These recommendations are a challenge in veterinary clinical pathology, especially when only small numbers of reference individuals are available.