Abstract: Effects of F-strain Mycoplasma gallisepticum (FMG) inoculation and 1.5% supplemental dietary Poultry Fat (PF) on the blood characteristics of commercial layers between 24 and 58 wk of age were investigated. Sham and FMG inoculations were administered at 12 (before lay) and 22 (early in lay) wk of age and dietary treatments [Basal Control Diets (BC) and basal control diets with 1.5% supplemental PF (BCPF)] were initiated at 20 wk of age. In birds inoculated at 12 wk of age (sham or FMG), total plasma protein and serum cholesterol concentrations were increased when the BCPF rather than the BC diet was fed and in birds fed, BC diets, inoculation at 22 rather than at 12 wk of age (sham or FMG) increased total plasma protein and serum cholesterol concentrations. At wk 34, serum triglyceride concentrations were reduced in birds inoculated with FMG (12 or 22 wk of age) in comparison to sham-inoculated controls. Furthermore, at wk 34, serum calcium concentrations were greater in birds that had been sham-inoculated on wk 22 in comparison to those inoculated with FMG at the same time or to those that were sham-or FMG-inoculated on wk 12. Conversely, on wk 58, serum calcium concentrations were greater in birds that had been inoculated with FMG at 22 wk in comparison to those that had been sham-inoculated at 12 or 22 wk of age. In conclusion, serum calcium concentrations may be decreased during peak egg production by a prelay inoculation procedure or the specific inoculation of FMG at the onset of lay; whereas calcium levels may become elevated late in lay by an FMG inoculation at lay onset. Also, while the inoculation of FMG may reduce serum triglyceride levels in layers during peak egg production, 1.5% supplemental dietary PF may interact with the age (12 or 22 wk) at which an inoculation (sham or FMG) is given to influence serum cholesterol and total plasma protein levels in layers.