Introduction: ¹³C, ¹⁸F and ¹²³I fatty acids (FA) are used for myocardial imaging. Recently, our group showed that [^99mTc]-labeled "4+1" FA are extracted into the rat and guinea pig myocardium. The present study evaluates determinants of myocardial uptake and whole body biodistribution of these FA derivatives. Methods: Studies were performed with isolated perfused hearts of Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) with a FAT/CD36 deficiency, as well as with heart type FA binding protein knockout mice (H-FABP)^-/- and H-FABP^+/+. Eight 4+1-^99mTc-FA were applied for 3 min followed by 1-min washout. A mathematical model was used to analyze FA dynamics and binding to proteins. Whole-body distribution was studied in rats with and without Tween 80. In vitro fractionation studies with [^99mTc]-FA assessed red blood cell uptake as well as association with plasma lipoproteins very low-density lipoprotein (VLDL), low-density lipoprotein (LDL) and high-density lipoprotein (HDL). Results: Myocardial extraction was 19.0-33.0% of the infused dose in isolated WKY and 15.2-26.4% in SHR hearts. However, H-FABP^-/- showed a marked reduction of tracer extraction [2.8±0.6%ID (percent injected dose) vs. 17±2%ID P<.001]. Uptake in red blood cells (<1.2%ID) and incorporation into lipoproteins were negligible. Incubation of ^99mTc-FA with albumin reduced ventricular extraction (P<.001) into the range of established iodinated FA tracers. polyoxyethylene(20) sorbitan monooleate improved the heart-to-liver ratio in the biodistribution studies. Conclusions: Myocardial uptake of [^99mTc]-FA 4+1 derivatives is dependent on H-FABP. These substances may therefore provide a new tool to specifically assess regional myocardial changes of H-FABP.