The adherence of 44 clinical isolates of Enterococcus faecalis, a common cause of endocarditis, and 13 Enterococcus faecium to substrates of six extracellular matrix (ECM) proteins was examined using ³⁵S-labeled bacteria. One E. faecalis strain, isolated from a patient with endocarditis, adhered to collagen types I and IV and another E. faecalis strain adhered to laminin and to collagen types I and IV. However, most isolates showed little adherence (<5% of added cells adhered) when grown at 37°C regardless of their source (endocarditis, urine or fecal sample). When grown at 46°C (but not when grown in CO₂ or nutrient limited media), most isolates of E. faecalis increased their adherence to immobilized laminin, collagen types I and IV but not to fibronectin, fibrinogen or bovine serum albumin, whereas none of the E. faecium increased adherence when grown at 46°C or 50°C. The adherence of E. faecalis was eliminated by digestion with trypsin, suggesting that a protein is somehow important, directly or indirectly, for adherence to occur. Pre-incubation of bacteria with soluble collagen types I and IV inhibited the adherence to these ECM proteins. These results demonstrate that in E. faecalis, adherence to ECM proteins is produced during routine in vitro growth conditions by occasional isolates and can be produced during certain stressful growth conditions by others. Whether this adherence relates to the propensity of E. faecalis to cause endocarditis remains to be determined.