The effects of 6/85-strain Mycoplasma gallisepticum (6/85MG) inoculation alone or in conjunction with F-strain M. gallisepticum (FMG) overlays and their timing on the blood characteristics of commercial egg-laying hens were investigated. Control birds received sham inoculations at 10 wk of age. Birds in a second treatment group were inoculated with 6/85MG at 10 wk of age, those in a third treatment group were inoculated with 6/85MG at 10 wk followed by an overlay inoculation of FMG at 22 wk, and those in a fourth treatment group were inoculated with 6/85MG at 10 wk followed by an overlay inoculation of FMG at 45 wk. Parameters investigated at 24, 32, 43, and 47 wk were hematocrit, plasma total protein, and serum calcium, triglycerides, and cholesterol. No significant treatment effects were noted for hematocrit, serum triglycerides, or serum cholesterol. However, at wk 32, plasma protein was greater in birds that received 6/85MG at 10 wk or 6/85MG at 10 wk and FMG at 22 wk in comparison to controls. Also, at wk 47, serum calcium concentration was greater in birds that received 6/85MG at 10 wk and FMG at 45 wk compared with controls and those that received 6/85MG at 10 wk and FMG at 22 wk. These results suggest that the prelay inoculation of pullets with 6/85MG may subsequently elevate plasma protein, and in conjunction with an FMG overlay at 45 wk, may increase serum calcium concentrations in laying hens.