Our nuclear pharmacy noted that 99mTc-sestamibi kits sometimes failed radiochemical purity (RCP) testing (i.e., RCP < 90%). All failed kits had been prepared with eluate from a newly arrived generator (ingrowth time > or = 72 h) which had been eluted > or = 6 h before the kit failure. The effects of 99mTc activity and eluate volume were then investigated to help explain the reason for the low RCP values. Our results demonstrated that higher failure rates of the 99mTc-sestamibi kits were noted when higher activities of 99mTc eluates were added, and the higher failure rates of the kits were associated with lower RCP values. In addition, higher kit failure rate and lower RCP values of the 5.55-GBq kits at 12 h postelution in comparison with the 11.1-GBq kits at 6 h (same eluate volume) indicated that the 99mTc activity and volume had a less detrimental effect on the 99mTc-sestamibi kit preparation than the 99mTc eluate age. The kit failures might be explained by the higher amount of 99mTc and the production of the free radicals during the long ingrowth time prior to generator elution. In conclusion, the use of a first elution from a long-ingrowth time generator at a later stage (i.e., 11.1 GBq at 6 h and 5.55 GBq at 12 h postelution) in preparation of a 99mTc-sestamibi kit is associated with a high rate of kit failure and should therefore be avoided.