Macrophages display different phenotypes that can switch in response to their micro-environment. In our earlier study (Chiang, C. S., Liu, W. C. and Jung, S. M., 2005. Compartmental responses after thoracic irradiation of mice: strain differences. Int. J. Radiat. Oncol. Biol. Phys. 62:862) on radiation-induced cytokine expression in lung lavage samples, there was a suggestion that the procedures used to harvest lung macrophages affected the profiles they expressed. To further explore this issue, we examined gene expression by cell populations, mainly macrophages, isolated by lavage from lung and peritoneal cavity following either in vivo or in vitro stimulation with LPS, IFN-gamma or irradiation. We found that expression of mRNA for tumor necrosis factor-alpha, IL-1 alpha/beta and IL-6 varied several fold depending on whether the assay was performed on cells immediately after isolation or after in vitro manipulation. The relative level of inducible nitric oxide synthase (iNOS) to arginase I (Arg I), which is frequently used as index of the M1 versus M2 functional macrophage phenotype, also varied. LPS stimulation in vivo was able to change the profile from Arg I expression to one where the iNOS pathway became dominant, but was unable to do this in vitro. This contrasts with the ability of IFN-gamma to generate an iNOS-dominant pathway in vitro, but not in vivo. This study cautions that the expression of inflammatory cytokines and the iNOS to Arg I ratio, which is often used as an index of their functional capacity, varies with the experimental conditions.