Clinical pain from several diseases has been reported to peak at different times along the day. This diverse reactivity to pain could be due to different contributions of the perception of (threshold) and reaction to (tolerance) pain to the pain perceived in various clinical conditions. To gain more insight into this subject we studied the mice hot plate licking and jumping responses, as indicative of pain threshold and pain tolerance, respectively. Both responses were measured every two hours for 24 h, and rhythmometrically analysed in groups of mice maintained for 21 days: (1) under different light-dark cycles (LD 12:12, LD 1:23 and LD 23:1) and tested in the absence or presence of naloxone (1 mg/kg, s.c.); (2) under LD 12:12 and tested in March and September; and, (3) under LD 12:12 and tested after being injected for 21 days with amitriptyline (10 mg/kg/day, i.p.). The main findings were: (1) the co-existence of two distinct diurnal rhythms for licking and jumping responses, whose phases differed by 6 hours under LD 12:12; (2) both rhythms were disrupted or considerably weakened under LD 23:1 and LD 1:23; (3) the endogenous opioid system seems to be involved in suppressing both diurnal rhythms under LD 23:1; (4) the mesor and amplitude, but not the acrophase, of the jumping rhythm seem to be seasonally modulated by opioid influences; and (5) chronic amitriptyline administration induced a significant phase delay of the diurnal rhythm for licking and abolished that for jumping. The data suggest that pain threshold and pain tolerance follow different diurnal rhythms, which might be important for explaining the diverse 24 hour time courses of the reactivity to painful clinical affections.