We have obtained transformants of Streptococcus parasanguis FW213 containing allelic replacements in several chromosomal loci. Transformation occurred following electroporation with nonreplicating plasmids carrying two antibiotic-resistance-encoding genes, one of which is inserted into DNA homologous to the chromosomal target. In contrast with other streptococci, S. parasanguis FW213 is not transformed by linear DNA. Mutations in nonreplicating plasmid DNA preferentially replaced their homologues in the S. parasanguis FW213 chromosome by a double-crossover homologous recombination event, as shown by the fact that over 90% of transformants were sensitive to the vector-coded antibiotic marker. Southern blot analysis of these transformants showed that three of the five target loci had been mutated, and that the wild-type sequence had been replaced by the mutated sequence carried on the transforming plasmid. This bias toward homologous replacement rather than integration of the entire transforming plasmid DNA simplifies site-specific mutagenesis and genetic analysis of the streptococcal chromosome.