The vertical distribution of thermotolerant (37 degrees C and 45 degrees C) free-living amoebae (FLA) in warm monomictic lakes was determined in relation to the onset of thermal stratification and associated physical and chemical changes. The position of abiotic or biotic particulate layers in the water column was located by using a submersible horizontal beam transmissometer that measures attenuance, or the absorption and scattering of light by particulates in the water column. During mixis, the vertical distribution of amoebae was sporadic with significant numbers of FLA only occurring in clay layers caused by runoff after heavy rains. With the onset of thermal stratification in the lakes, phytoplankton layers began to form. Few amoebae were isolated from layers containing flagellated phytoplankton; however, significant (P less than 0.005) numbers of FLA were isolated from two particulate layers dominated by the filamentous blue-green algae Aphanizomenon and Lyngbya, respectively. By late June, a persistent detrital or decomposition layer formed in the lower metalimnion, as well as a hypolimnetic iron layer where the Fe2+ state was predominant. In this midsummer period, 13 Naegleria fowleri were isolated, with three from the detrital layer and seven from the iron layer. The presence of attenuation zones was found to be the best indicator of the vertical distribution of FLA in the water column, and such layers represent an important, previously undescribed habitat for potentially pathogenic FLA.