Summary: Two in vitro assays are described that can distinguish between two modes of attachment of Azospirillum brasilense Sp7 to wheat roots, and quantify the number of attached bacteria. The first assay measures A. brasilense adsorption (Ads), which reaches a maximal level within 2 h of incubation. Adsorption forces are weak, since adsorbed bacteria can be quantitatively removed by vortexing the root in water. In addition, adsorption is strongly reduced by pretreatment of A. brasilense cells with pronase E. The second assay measures A. brasilense anchoring (Anc), which begins only after 8 h of incubation and reaches a maximal level after 16 h. That adsorption and anchoring are different phenomena is further demonstrated by the properties of two classes ofA. brasilense attachment mutants. The first class of mutants, deficient in the production of a particular surface polysaccharide, has completely lost anchoring capability, but maintains wild-type adsorption capacity (Ads+Anc-). Mutants of the second class are defective in adsorption, but not in anchoring (Ads-Anc+). On the basis of these data, a two-step attachment mechanism of A. brasilense to wheat roots is proposed. The first step consists of a rapid and weak adsorption and depends on bacterial surface protein; the second step is, at least in vitro, independent of the first and consists of firm anchoring of adsorbed and free bacteria by means of bacterial extracellular polysaccharide.