Rapamycin is a drug with several important clinical uses. Its complex structure means that total synthesis of this natural product and its analogues is demanding and lengthy. A more expeditious approach is to utilise biosynthesis to enable the generation of otherwise synthetically intractable analogues. In order to achieve this, rules governing biosynthetic precursor substrate preference must be established. Through determining these rules and synthesising and administering suitable substrate precursors, we demonstrate the first generation of fluorinated rapamycin analogues. Here we report the generation of six new fluororapamycins.