The protozoan parasite Toxoplasma gondii infects a wide range of vertebrate hosts and is an important opportunistic pathogen in immunocompromised humans. Although Toxoplasma is amenable to both biochemical and cellular experimental approaches, the molecular basis of its success as an intracellular parasite is poorly understood. To provide a system for molecular genetic analyses, we have developed a stable DNA transformation system for Toxoplasma based on complementation of its naturally occurring tryptophan auxotrophy. Complementation was accomplished by expressing the Escherichia coli trpB gene, encoding the beta subunit of tryptophan synthase (EC 4.2.1.20), the enzyme that catalyzes the formation of tryptophan from indole plus serine. Transformants were obtained by electroporation of a plasmid, called SAG1/trpB, containing the trpB gene flanked by Toxoplasma surface antigen 1 (SAG1) gene sequences and selection for growth on indole. Transformants were obtained with circular forms of the SAG1/trpB plasmid with efficiencies of 10(-4) per cell. Transformation with either circular or linear SAG1/trpB resulted in integration into the genome at distinct, nonhomologous sites. Trp+ transformants typically contained tandemly repeated copies of the SAG1/trpB plasmid and were stable in the absence of continued selection. The Trp phenotype provides a dominant selectable marker that should allow expression of foreign or altered genes in Toxoplasma and facilitate molecular analyses of genes important for intracellular survival.