Streptococcus gordonii DL1 (Challis) bears coaggregation-mediating surface adhesins which recognize galactoside-containing surface polysaccharides on Streptococcus oralis 34, Streptococcus oralis C104, and Streptococcus SM PK509. Fifty-nine spontaneously-occurring coaggregation-defective (Cog-) mutants of S. gordonii DL1 unable to coaggregate with partner streptococci were isolated. Six representative Cog- mutants were characterized by their coaggregation properties with four Actinomyces naeslundii strains (T14V, PK947, PK606, PK984), Veillonella atypica PK1910, and Propionibacterium acnes PK93. The six representative Cog- mutants showed altered coaggregation with their streptococcal partners, A. naeslundii PK947, and P. acnes PK93. Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation between S. gordonii DL1 and its partner bacteria is proposed. The potential use of these mutants in studies of oral biofilms is discussed.