Reference materials (RMs) for Listeria monocytogenes were developed and tested for their suitability in three European collaborative studies. In the first study, the 40 participants, each tested 20 RMs. In the second, the 42 participants each tested 25 RMs in combination with RMs containing competitive micro-organisms. In the third study, the 30 participants each tested a variable number of RMs in combination with food products. In all studies, RMs with c. five colony forming units (cfu) per capsule were used. In the third study RMs at two levels were used containing c. 5 and c. 100 cfu per capsule. Based on the number of cfu per capsule or the fraction of RMs not containing L. monocytogenes, the intended isolation frequency was calculated. All participants tested the materials according to their own procedures. These non-standard methods (NSMs) were divisible into six groups of comparable methods. In the first and second study a standardized method was also used by each participant. The results from the first study showed that most laboratories were able to isolate L. monocytogenes in the expected isolation frequencies. Using the standardized method 97% of the RMs were found positive for Listeria. In the second study only c. 80% of the RMs were found positive as a result of the addition of the competitors (Enterococcus faecium, Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis at a level of, respectively, 8 × 10³, 11 × 10³, 5 × 10³ and 5 × 10³ cfu per capsule). The groups of NSM methods based on the Food and Drug Administration's method for the detection of L. monocytogenes seemed to be less effected by the addition of the competitors than the groups of NSM methods based on the US Department of Agriculture's method. Addition of RMs to food products resulted in a substantial decrease in the isolation of L. monocytogenes. No relationship was apparent between the type of method used and the type of food product tested. It was concluded that the RMs are suitable for testing laboratory performance of the detection methods for L. monocytogenes.