The formalin test is a well-established model for assessing nociceptive processes and analgesic drug effects. Previous studies have provided statistical validation of optimal procedures for conducting and scoring the rat formalin test. In the mouse model, formalin concentration has been subjected to validation studies. The present research extended previous work by subjecting two additional parameters - the behaviors that should be scored and the optimal interval for second-phase formalin response - to empirical validation. Five behavioral (formalin-induced favoring, lifting, and biting/licking, rearing and locomotion) and two physiological measures (paw weight and paw thickness increases reflective of formalin-induced inflammation) were examined under four formalin concentrations (Exp. 1: 0.5, 1.0, 5.0, and 10.0%/25µl formalin) or under four morphine doses (Exp. 2: 0.0, 1.0, 5.0, and 10.0 mg/kg, s.c. with 5% formalin concentration). Multiple regression analyses defined the optimal second-phase formalin response in outbred, Swiss-Webster mice as 15-35 min post formalin injection, and revealed that the second-phase response is best characterized by the cumulative time spent biting/licking the injected paw. Finally, paw physiological measures provided convergent evidence of nociceptive and antinociceptive processes in the mouse formalin test. Copyright 1998European Federation of Chapters of the International Association for the Study of Pain.