Twenty-four 70-week-old and sixteen 27-week-old white leghorn hens were challenged with R strain Mycoplasma gallisepticum (MG) by injection into the caudal thoracic air sac and infraorbital sinus. Eggs were collected daily and cultured within 7 days or incubated for 18 days. Vitelline membranes of eggs were cultured directly; in 18-day-old embryos, cultures were taken from the yolk sac, air sacs, and oral cavity. Culture of vitelline membrane of eggs within 2 days was compared with culture of eggs stored 10 days post oviposition. The first MG-positive egg was laid 2 days postinfection (PI). Hens continued to lay positive eggs to the end of the experiments. There was no significant difference in MG recovery between eggs cultured within 2 days and those cultured 10 days post oviposition. MG was isolated at a significantly higher rate from eggs than from 18-day-old embryos. MG was isolated at a higher rate from the yolk sac of 18-day-old embryos than from the air sacs or oral cavity of the same embryos.