The formalin test is used as a primary behavioural screen for assaying the antinociceptive activity of compounds in laboratory rodents. After hindpaw formalin injection, nociceptive behaviours are expressed in a biphasic pattern and correlate closely with the concentration of formalin injected. Here, the antinociceptive efficacy of six compounds used in the clinical treatment of chronic pain was compared in rats injected with either 1% or 5% formalin. Predictably, the anti-inflammatory drug ibuprofen (30-300 mg/kg) attenuated second phase (16-40 min) nociceptive behaviours in 5% formalin-injected rats; 1% formalin-injected rats were unaffected. The anti-epileptic drugs gabapentin (50-200 mg/kg) and lamotrigine (10-60 mg/kg) were antinociceptive only during second phase in both 1% and 5% tests. Notably, they were 2-4 times more potent in 1% vs 5% formalin-injected rats. The dual monoamine reuptake inhibitor duloxetine (3-60 mg/kg) consistently attenuated nociceptive behaviours during first phase and interphase in both tests. However second phase nociception was only attenuated in 5% formalin-injected rats giving rise to a 6 fold increase in potency compared with 1% formalin-injected rats. The micro-opioid receptor agonist morphine (1-6 mg/kg) and the combined micro-opioid receptor agonist/monoamine reuptake inhibitor tramadol (5-50 mg/kg) both attenuated nociceptive behaviours throughout the duration of both the 1% and 5% tests; no difference in antinociceptive potency occurred for either compound during second phase. Thus, for mechanistically-distinct compounds the predictive antinociceptive capacity of the formalin test can vary with the concentration of formalin injected, likely as a result of peripheral and central nociceptive signalling mechanisms being differentially engaged.