Virulent strains of Mycoplasma pneumoniae, PI-1428 and M129, were radiolabeled wtih [3H]palmitic acid or [3H]thymidine and examined for attachment to hamster tracheal organ cultures, tracheal outgrowth monolayers, human O-positive erythrocytes, and human WiDr carcinoma cell cultures. Although attachment to each cell substrate was readily detected, the WiDr cell culture monolayers provided the most satisfactory substrate for quantitating mycoplasma attachment. Serious technical limitations were encountered with each of the other substrates that we examined; these limitations interfered with reproducibility or sensitivity and rendered tracheal organ cultures and erythrocyte suspensions unsuitable for routine attachment and attachment inhibition assays. Moreover, the WiDr cell monolayer was the most sensitive substrate for determining attachment inhibition activity in protein-containing extracts prepared from M. pneumoniae. The significance of these findings is discussed.