The intestinal lesions of Mycobacterium avium subspecies paratuberculosis (M. a. ptb) infection are characterized by extensive granulomatous lesions with high bacterial burdens. The objective of this study was to examine macrophage activation phenotype and pro-inflammatory cytokine expression specifically within intestinal granulomatous lesions from animals naturally infected with M. a. ptb. To test the hypothesis that granuloma macrophages have a non-activated phenotype, we examined granulomatous lesions from the ileocecal valve of cattle in the clinical stages of Johne's disease. We determined protein expression by immunohistochemistry and compared this to gene expression determined by quantitative reverse transcriptase real time polymerase chain reaction (Q-RT-PCR). Laser capture microdissection was used to recover granuloma cells from intestinal lesions, thereby allowing for direct sampling from the granuloma microenvironment. Our immunohistochemical studies included stains to detect mycobacterial antigen, iNOS as a marker of macrophage activation, and IFN-γ. Using laser capture microdissection, granuloma cells were collected onto a thermoplastic film held on a collection cap, (CapSure® HS LCM Caps). We collected roughly one 80% confluent cap per experiment. We measured gene expression of iNOS and IFN-γ in the captured cells by Q-RT-PCR using the Syber green system. The results of this study demonstrate that most granulomatous lesions had high bacterial burdens. Protein expression of iNOS was low within the granulomatous foci. Q-RT-PCR demonstrated variable IFN-γ gene expression within granulomas and surrounding lamina propria. Gene expression for iNOS within granuloma cells was low. These data suggest that macrophages in intestinal granulomatous lesions of bovine Johne's disease do not have strong iNOS expression, suggesting a non-activated phenotype.