Numerous studies have explored the effects of morphine on cognitive function, while knowledge concerning methadone is scarce. This study is the first in line, being part of a newly started project. The aim of this study was to examine whether long-term administration of methadone results in deficits in performance in the Novelty-test and changes in phosphorylation of calcium/calmodulin kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) in the hippocampus. Rats were administrated (sc) methadone, morphine or saline once per day for three weeks (2.5-10 mg/kg), and novelty-tested one hour and one week after the last injection. Western blotting was performed on homogenate from hippocampus, taken out directly after the novelty-testing, to detect levels of phosphorylated CaMKII (p-CaMKII) and ERK (p-ERK). p-CaMKII was significantly down-regulated one week after the last injection of methadone, while no significant difference in p-ERK was observed. There were no significant differences between the groups in the novelty-test one week after the last injection, while one hour after the last injection the animals were to affected by the methadone to perform the test. Our findings indicate that long-term methadone exposure may specifically affect protein phosphorylation, but don’t seem to have a persistent effect on novelty-exploration. This may also be due to the Novelty-test not being sensitive enough to pick up small differences between the exposed groups and the control group.