A previous study from our laboratory on F-strain Mycoplasma gallisepticum-inoculated layers showed a significant increase in arterial partial pressure of oxygen (pO(2)), which is generally associated with an oxygen-dependent improvement in tissue oxygenation. The aim of this study was to determine whether a killed (bacterin) and live TS-11-strain M. gallisepticum (TS-11-MG) vaccine treatment combination could further enhance the arterial pO(2) levels in layer chickens. The experiment was conducted in 2 trials and arranged in a completely randomized experimental design with 4 treatments. The treatments consisted of a control M. gallisepticum, bacterin, TS-11-MG, and bacterin + TS-11-MG combined, with all treatments receiving the R low strain of MG at 30 wk of age (WOA). In each of the 2 trials, 160 one-day-old MG-free pullets were raised to 10 WOA and were transported to a poultry disease isolation facility. Sixteen isolation units were divided into 4 treatment groups, and each of the 4 treatment groups had 4 replication units, with 10 birds/unit (40 birds/treatment). Venous blood samples were collected at the termination of the study at 56 WOA. The TS-11-MG-vaccinated chickens had a higher (P ≤ 0.05) blood pO(2) and a lower (P ≤ 0.05) partial pressure of CO(2) when compared with the control and combined MG-vaccinated groups. However, no significant blood pO(2) differences were observed between the bacterin and TS-11-MG treatment groups. Hematocrit and blood concentrations of hemoglobin were not statistically different among treatments, but were numerically higher in the TS-11-MG treatment group. There was a significant (P ≤ 0.05) treatment effect on blood concentrations of Na(+), Ca(2+), and anion, but no significant effect on glucose, cholesterol, triglyceride, or osmolality. These data suggest that the inoculation of layers with TS-11-MG was more effective in elevating pO(2) than was inoculation with TS-11-MG + bacterin combined.