The aim of this study was to verify the most suitable vehicle and concentration for testing Euxyl K 400 and its individual ingredients, and to evaluate the prevalence of sensitization to this preservative over the years in Italy. From January 1991 to October 1994, Euxyl K 2.5% pet. was positive in 99 patients (35 male, 64 female) out of 3455 (2.8%). Of these, 22 out of 855 patients had a positive reaction during 1991 (2.6%), 29 out of 1037 in 1992 (2.8%), 28 out of 858 in 1993 (3.3%), and 20 out of 705 in 1994 (2.8%). 51 of the 99 patients with a reaction to Euxyl K 400 2.5% pet. showed a positive reaction to dibromodicyanobutane 0.5% pet. and 2 to phenoxyethanol 5% pet. The results of patch testing with serial dilutions of Euxyl K 400 in different vehicles indicate that water is a good vehicle for testing the preservative. However, since Euxyl K 400 is only hydrosoluble to a limited extent, the maximum concentration that can be tested using water is 0.5%, and so with this concentration about 40% of sensitized patients are missed. The results of patch testing with serial dilutions of Euxyl K 400 in petrolatum demonstrate that concentrations lower than 2.5% are not suitable for detection of all sensitized patients. Euxyl K 400 in ethanol frequently causes irritant reactions without offering significant advantages in detecting sensitized patients.