Several proteins with NAD+:arginine ADP-ribosyltransferase (ART) activity are expressed in T cells and affect their function. Rat T cells that express the ART designated RT6 are determinants of the expression of autoimmune diabetes. In the mouse, a 35-kDa ecto-ART modulates the proliferation and functional activity of CTL. Here we report on mouse ARTs designated Rt6-1 and Rt6-2 in BALB/c and C57BL/6 mice. mRNAs for Rt6-1 and Rt6-2 were found in spleen, thymus, and intestinal tissue of both strains, but Rt6-1 mRNA in C57BL/6 mice was detected only at low levels. Rt6-1 and Rt6-2 cDNAs from both strains were cloned and sequenced. Predicted amino acid sequences of Rt6-2 were identical in both strains, but there was an in-frame stop codon in the sequence of Rt6-1 in C57BL/6 mice not present in BALB/c mice. Recombinant C57BL/6 Rt6-2 and BALB/c Rt6-1 proteins expressed in COS1 cells exhibited ART activity and were documented to be glycosylphosphatidylinositol-linked membrane proteins. COS-1 cells transfected with a C57BL/6 Rt6-1 cDNA construct expressed a truncated protein consistent in size with that predicted by the presence of the stop codon. This approximately 21-kDa protein appeared not to be glycosylphosphatidylinositol linked to the cell surface and lacked ART activity. C57BL/6 Rt6-1 therefore appears to be a naturally occurring ART knockout. The expression of Rt6-1 and Rt6-2 mRNAs in lymphoid tissues suggests that these ARTs may regulate immune system functions. Expression of Rt6-2 or another redundant ART may compensate for the lack of enzymatically active Rt6-1 in C57BL/6 mice.