Fungal highly reducing polyketide synthases (hrPKSs) are remarkable multidomain enzymes that catalyse the biosynthesis of a diverse range of structurally complex compounds. During biosynthesis, the ketosynthase (KS) and acyltransferase (AT) domains of the condensing region are visited by the acyl carrier protein (ACP) domain during every cycle, catalysing chain priming and elongation reactions. Despite their significance, our comprehension of how these steps contribute to biosynthetic fidelity remains poorly understood. Herein, we report the full in vitro reconstitution of each catalytic step carried out by a hrPKS condensing region. Application of intact protein mass spectrometry shows that the AT domain operates selective acyl transfer to the ACP domain, ensuring loading of the correct starter / extender units, which is governed by a substrate recycling mechanism and kinetic parameters towards each substrate. The KS domain is shown to select for the correct starter unit for transacylation and subsequent chain elongation, yet harbours flexibility towards the extender unit supplied. This work provides the first comprehensive interrogation of a fungal hrPKS condensing region, which both affirms and expands our understanding of how these systems function, and provides a framework for future bioengineering efforts.