The reliability of isoelectric focusing (IEF) of sarcoplasmic proteins for fish species identification was evaluated by a collaborative study among eight European laboratories. Each laboratory used its own method of IEF to identify 10 unknown samples of raw muscle by means of reference material. In 93% of cases the assignment between sample and reference was correct. In a second study, the influence of extradant (water, low ionic strength buffer, or detergent) and the position of sample application on the protein pattern was examined. Working with light muscle of rainbow trout (Oncorhynchus mykiss), it was found that the type of extractant did not influence the protein pattern. Comparison of the patterns of samples, which had been applied near the anode, in the middle, or near the cathode, revealed differences in the number and position of the protein bands under the experimental conditions applied by most laboratories. This effect was not observed with the Phast System.