Highly selective, positive allosteric modulators (PAMs) of the M subtype of muscarinic acetylcholine receptor have emerged as an exciting new approach to potentially improve cognitive function in patients suffering from Alzheimer's disease and schizophrenia. Discovery programs have produced a structurally diverse range of M receptor PAMs with distinct pharmacological properties, including different extents of agonist activity and differences in signal bias. This includes biased M receptor PAMs that can potentiate coupling of the receptor to activation of phospholipase C (PLC) but not phospholipase D (PLD). However, little is known about the role of PLD in M receptor signaling in native systems, and it is not clear whether biased M PAMs display differences in modulating M-mediated responses in native tissue. Using PLD inhibitors and PLD knockout mice, we showed that PLD was necessary for the induction of M-dependent long-term depression (LTD) in the prefrontal cortex (PFC). Furthermore, biased M PAMs that did not couple to PLD not only failed to potentiate orthosteric agonist-induced LTD but also blocked M-dependent LTD in the PFC. In contrast, biased and nonbiased M PAMs acted similarly in potentiating M-dependent electrophysiological responses that were PLD independent. These findings demonstrate that PLD plays a critical role in the ability of M PAMs to modulate certain central nervous system (CNS) functions and that biased M PAMs function differently in brain regions implicated in cognition.