The stimulation of human gamma delta T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1-4. Among these latter, TUBag4 has already been biochemically characterized as a gamma-X derivative of 5'-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fournié, J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5'-UTP nucleotide moiety. On selected V gamma 9/V delta 2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of gamma delta PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized gamma-isopentenyl 5'-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human gamma delta T cells (Tanaka, Y., Morita, C.T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells.