The biologic and microenvironmental factors determining (99m)Tc sestamibi (MIBI) and (99m)Tc tetrofosmin (TF) uptake in breast tumors are incompletely understood, especially in P-glycoprotein (Pgp)-negative tumors. We analyzed the influence of glucose administration on the uptake and retention of MIBI and TF in Pgp-negative tumor-bearing mice in vivo. Twenty (20) mice bearing Ehrlich ascites tumor cell (EATC) xenografts were divided into four groups: (1) MIBI, (2) MIBI+glucose, (3) TF, and (4) TF+glucose. Glucose was administered (5.0 g/kg body weight) intraperitoreally (i.p.) 1 hour before scintigraphy. There were significant differences between the E-UPR MIBI and MIBI+glucose groups (p = 0.009) and minor differences in L-UPR between these groups (p = 0.04). There was a significant inverse correlation between E-UPR of MIBI and glucose levels (r = 0.71, p = 0.02). Comparing the four groups, the highest E-UPR was obtained in the MIBI group (p = 0.006). Other parameters were not different in the MIBI and MIBI+glucose groups and in the TF and TF+glucose groups. Increased blood glucose level affected the MIBI uptake of tumor tissue, particularly for E-UPR. We suggest that these findings were due to basically decreased blood flow and secondarily decreased extracellular pH. However, glucose administration did not affect TF.