Technical Abstract: The use of luminescent technology may serve as a viable model for the real-time validation of various pre-harvest interventions on the colonization or shedding of Escherichia coli O157:H7 within cattle. The objective of this study was to determine if the growth of E. coli O157:H7 (ATCC 43888) in rumen and fecal fluid is altered when transformed with one of the two luminescent plasmids: pAK1-lux or pXEN-13. Growths of all three variants were analyzed to verify that all three strains grow similarly under standard laboratory medium conditions. Strains harboring the luminescent plasmids were grown in comparison to the non-transformed parental strain in rumen and fecal fluid media for 8 h in replicates of three. All three variants grew similarly in the absence of antibiotic pressure, and the plasmids grew similarly in the presence of ampicillin pressure. There was no treatment by time interaction (P < 0.36) observed within rumen fluid media based off log10 values and no treatment by time interaction (P < 0.78) in percent viability from 0 h observed. In the log10 analysis of fecal fluid media, there was a treatment effect (P < 0.001), a time effect (P < 0.001), and a treatment by time interaction (P < 0.006). In percent viability from 0 h, there was no treatment by time interaction (P < 0.67) observed within fecal fluid media. The RLU (reflective light units; photon/pixel second) emissions of the plasmids within rumen fluid exhibited a treatment by time interaction (P < 0.003) and a treatment by time interaction (P < 0.001) within fecal fluid media. Growth of both E. coli O157:H7 transformed with the biophotonic plasmids were not altered in comparison to the WT, suggesting that both plasmids may serve as valid models for in vivo studies.