In order to evaluate the effects of formalin concentration and inhalational anesthetics in formalin tests, we injected 5 concentrations of formalin into awake and anesthetized rats and investigated their behavior and the c-fos immunoreactivity of their spinal cords.Sixty adult male Sprague-Dawley rats were divided into 10 experimental groups. Each rat was either manually restrained or subdued through several minutes of 2% to 2.5% halothane inhalation, and then 1 of 5 concentrations of 0%, 5%, 10%, 27%, or 100% formalin (0%, 1.85%, 3.7%, 10%, or 37% formaldehyde solutions) was injected into its rear paw. Nociceptive behaviors were checked for 1 hour, 6 times for each 5-minute period. Two hours after the formalin injections, the rats were killed and c-fos immunoreactivity was measured.Typical responses were observed in the 5% and 10% formalin (1.85% and 3.7% formaldehyde) groups, and nociceptive behaviors were lower in the 27% and 100% formalin (10% and 37% formaldehyde) groups. The number of c-fos-positive cells increased as the formalin concentration increased. Halothane inhalation affected the results of both the behavior and the c-fos immunoreactivity, especially in the 10% formalin (3.7% formaldehyde) group.It is desirable to describe both formalin and formaldehyde concentrations simultaneously and to do formalin tests without inhalational anesthetics.