Opioids, such as morphine, can directly alter immune function via receptors expressed on immunocompetent cells. However, several studies have questioned the classical opioid nature of this change in immune response. Therefore, it is unclear how opioids that are not from the same structural class as morphine (4,5-epoxymorphinan), will modulate the immune system, if they do not behave in a classical opioid manner. Therefore, the aim of this study was to investigate the in vitro modulatory effects of a range of non-4,5-epoxymorphinan opioids on splenocyte proliferation and compare the response characteristics to their central opioid characteristics. The modulation of concanavalin A stimulated mouse splenocyte proliferation by non-4,5-epoxymorphinan opioids resulted in three types of responses: an inhibitory concentration-response curve (e.g. methadone, inhibitory EC(50)=79.4 microM), an inverted bell shaped curve (e.g. fentanyl, inhibitory EC(50)=0.06 microM) and an induction concentration response curve (e.g. nor-binaltorphimine, induction EC(50)=0.16 microM). Non-stereoselectivity, naloxone-insensitivity, naloxone-sensitivity and non-classical opioid rank order of effect were all observed. These data support the non-classical opioid nature of direct opioid modulation of splenocyte proliferation.