The behavioural response to formalin-induced persistent pain was examined in male and female rats both unfamiliar and familiar with the test apparatus. Rats were subcutaneously injected with 50 microliters of formalin (10%) in the hindpaw and placed in the test cage (60 min). Licking and Flexing duration and Paw-Jerk frequency were recorded. Licking and Flexing lasted longer in females than males, while Paw-Jerk occurred in both sexes with comparable frequencies. Flexing and Paw-Jerk were lower in animals unfamiliar with the test apparatus. Therefore, behavioural responses to pain appeared to be affected by sex and familiarization with the experimental setting in different and independent ways.