The effect of different formalin concentrations on the nociceptive response in the formalin test was examined in mice. Subcutaneous formalin injection induces 2 distinct periods of high licking activity: an early phase lasting the first 5 min, and a late phase lasting 20-30 min after the injection. Formalin concentrations of 0.02-0.2% induced only the early phase, while concentrations of 1% or more induced both the early phase and the late phase. The ability of the test to show the antinociceptive effect of morphine and acetylsalicylic acid was similar for high and low formalin concentrations. For both these analgesics, a lower dose was needed to induce antinociception in the late phase than in the early phase using the same formalin concentration. Indomethacin had no effect in the early phase. In the late phase indomethacin induced antinociception when 1% formalin was used, while no significant effect was observed using 5% formalin. Clear histological changes in the paw were demonstrated after formalin concentrations that induced both phases. Lower formalin concentrations induced only very small changes. Using a low formalin concentration (0.2%), repeated testing using the same paw could be performed at intervals of 1 week without any significant change in the response. It was concluded that the formalin concentration should be kept as low as possible to minimize the suffering of the animal. Formalin concentrations of 0.05-0.2% are recommended for studying the early phase. Formalin concentrations of 1% or higher have to be used when studying the nociceptive response in the late phase.