TPMT*16 is assigned no function based on moderate evidence. Erythrocytes from individuals genotyped TPMT*1/*16 exhibited intermediate enzymatic activity (15226673, 15652243, 33118454). In vitro studies found that TPMT.16 exhibited decreased affinity for 6-TG and enzymatic activity was decreased by 84% and 68% relative to TPMT.1 in mammalian (18708949) and yeast (15652243) cells, respectively. An in silico prediction model of TPMT variant functionality can predict >95% of variance when including TPMT*16 along with the most common variants TPMT*2, *3A, and *3C, compared to 70% without TPMT*16 (32973300). Five out of ten variant effect predictors tested by CPIC staff predict that the TPMT*16-defining SNV, c.488G>A (p.R163H), impairs protein function.