T cell clone 3Tx19 detects a Toxoplasma gondii tachyzoite protein which, in high resolution 2D gel electrophoresis, runs at 36 kDa apparent MW with two spots of pI 5.9 and 6.5, thus exhibiting a migration pattern distinct from those of other known Toxoplasma antigens. The sequences of peptide fragments from tryptic digestion of the more prominent protein spot allowed the design of oligonucleotide primers to obtain the coding cDNA sequence. Sequence analysis of cDNA from strain BK revealed a 363 amino acid open reading frame, defined by all nine peptide sequences determined. The deduced protein sequence contains two hydrophobic segments, one near the N-terminus including a predicted signal peptide and a shorter second at the carboxy terminus, but homology to any other known protein is lacking. With synthetic peptides covering the complete primary structure, the epitope for clone 3Tx19 was mapped within the deduced partial sequence, which had remained unconfirmed by tryptic peptides. Antibodies raised against another, putative B cell epitope peptide detected the same two protein spots in 2D gel, indicating that they are antigenically related isoforms. The protein p36 is expressed by T. gondii isolates of all three intraspecies subgroups, but not in the bradyzoite stage. In intracellular tachyzoites, p36 colocalizes with rhoptry proteins and has a distribution pattern disparate from that of dense granule and microneme proteins. Subcellular fractionation indicated that p36 is a soluble constituent of tachyzoites. We suggest that this T cell-stimulatory novel rhoptry protein of T. gondii be named ROP9. It represents a marker of the tachyzoite stage.