The interaction between Mycoplasma genitalium and human lung fibroblasts (HLFs) was studied with the use of wild-type and hemadsorption-negative (HA−) mycoplasmas. [35S]-methionine-labeled M. genitalium adhered to HLFs by first-order kinetics, with maximal interaction occurring at ∼2 hours. Electron microscopy of chemically fixed cells revealed an almost immediate association of mycoplasmas with the HLF plasma membrane that was mediated by the mycoplasma tip and a nap-like layer, which appeared to extend from the tip around much of the mycoplasmal unit membrane. Following cytadherence, M. genitalium appeared capable of invading the intracellular spaces of targeted HLF cells, possibly by receptor-mediated endocytosis. Spontaneously arising HA− variants of M. genitalium strain G37 failed to adhere to HLF cells and were distinguished on the basis of their protein profiles. SDS-PAGE analysis of the class I (lacking the 140-kd protein but containing a polypeptide doublet at ∼ 140-kd) and class II (lacking the 140-kd protein and doublet) variants of M. genitalium revealed that class I variants contain a doublet protein in the 140-kd region, which reacted with a monoclonal antibody generated to the adhesin-implicated 140-kd protein (P 140) of wild-type M. genitalium. Class II variants completely lacked the 140-kd protein or immunologically related peptides.