Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem,S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, bothclfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as theS. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in ≥80% of the rats (80% infective dose [ID₈₀]) with the parent lactococcus was ≥10⁷CFU. In contrast, clfA-expressing andfnbA-expressing lactococci required only 10⁵CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID₈₀ = 10⁴ to 10⁵ CFU) in this model. The results confirmed the role ofclfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of theclfA and fnbA products should be blocked for the therapy to be effective.