INTRODUCTION: In vivo pH imaging has been a field of interest for molecular imaging for many years. This is especially important for determining tumor acidity, an important driving force of tumor invasion and metastasis formation, but also in the process of apoptosis. METHODS: 2-(4-[(123)I]iodophenethyl)-2-methylmalonic acid (IPMM), 2-(4-[(123)I]iodophenethyl)-malonic acid (IPM), 2-(4-[(123)I]iodobenzyl)-malonic acid (IBMM) and 4-[(123)I]iodophthalic acid (IP) were radiolabeled via the Cu(+) isotopic nucleophilic exchange method. All tracers were tested in vitro in buffer systems to assess pH driven cell uptake. In vivo biodistribution of [(123)I]IPMM and [(123)I]IPM was determined in healthy mice and the pH targeting efficacy in vivo of [(123)I]IPM was evaluated in an anti-Fas monoclonal antibody (mAb) apoptosis model. In addition a mouse RIF-1 tumor model was explored in which tumor pH was decreased from 7.0 to 6.5 by means of induction of hyperglycemia in combination with administration of meta-iodobenzylguanidine. RESULTS: Radiosynthesis resulted in 15-20% for iodo-bromo exchange and 50-60% yield for iodo-iodo exchange while in vitro experiments showed a pH-sensitive uptake for all tracers. Shelf-life stability and in vivo stability was excellent for all tracers. [(123)I]IPMM and [(123)I]IPM showed a moderately fast predominantly biliary clearance while a high retention was observed in blood. The biodistribution profile of [(123)I]IPM was found to be most favorable in view of pH-specific imaging. [(123)I]IPM showed a clear pH-related uptake pattern in the RIF-1 tumor model. CONCLUSION: Iodine-123 labeled malonic acid derivates such as [(123)I]IPM show a clearly pH dependent uptake in tumor cells both in vitro and in vivo which allows to visualize regional acidosis. However, these compounds are not suitable for detection of apoptosis due to a poor acidosis effect.