Opioid‐induced respiratory depression (OIRD) is the primary cause of death from the overdose of opioid analgesics and drugs of abuse. We are interested in identifying molecular/cellular mechanisms underlying OIRD in central respiratory circuits, towards the aim of mitigating OIRD.Although multiple brainstem centers likely contribute to OIRD, we focused on the preBötzinger Complex (preBötC) since this locus is essential for generating rhythmic inspiratory drive. Using in vitro mouse brain slices containing the isolated preBötC, we confirmed that inspiratory burst frequency is depressed by bath‐applied DAMGO, a mu‐opioid receptor (MOR1) specific agonist. MOR1 is known to suppress excitability by signaling via the Gi/o pathway to suppress presynaptic calcium channels and activate GIRK K+ channels. The contribution of each effector pathway was tested with pharmacological and genetic tools. Unexpectedly, blockade of GIRK K+ channels (TertiapinQ) failed to rescue in vitro DAMGO‐depressed rhythms, and activation of GIRK channels (ML297) failed to mimic OIRD, suggesting a relatively modest role for MOR1‐mediated GIRK activation as a mechanism for OIRD. By contrast, slices derived from heterozygous CACNA1A (encoding P/Q‐type calcium channels) KO/+ animals were found to be 10‐fold sensitized to DAMGO depression, consistent with a major presynaptic component for OIRD. In addition, blockers of KCNQ K+ channels (XE991, Chromanol 293B) found in presynaptic terminals, rescued DAMGO‐depressed rhythms, and activators of KCNQ channels (ICA 69673, Retigabine) mimicked DAMGO‐mediated OIRD. Direct support for a presynaptic component of OIRD was provided by recording mEPSCs from genetically labelled Dbx1 neurons. Spontaneous excitatory mEPSCs frequency in TTX was suppressed by DAMGO (70% of cells) and restored by subsequent block of KCNQ channels (XE991) (~83% of cells), supporting the co‐localization of MOR1 and KCNQ K+ channels with P/Q‐type calcium channels to presynaptic terminals. In vivo plethysmography in unanesthetized animals combined with systemic injections with GIRK (ML297) and KCNQ (Retigabine) activators, along with morphine followed by KCNQ blocker (XE991), largely corroborated these in vitro findings. We hypothesize that preBötC OIRD is primarily due to MOR1‐mediated suppression of presynaptic calcium channels, modulated by co‐compartmentalized KCNQ K+ channels. By this model, decreased synaptic efficacy due to OIRD leads to collapse of the rhythmogenic capacity of the preBötC network. Although our results do not support a major role for GIRK K+ channels in OIRD, preliminary results suggest a secondary role for TWK‐type K+ leak channels in OIRD. Co‐expression of MOR1 and TREK2 in Xenopus oocytes produced currents that augmented ~2‐fold with DAMGO and blocked by fluoxetine. In vitro slice recordings revealed rescue of DAMGO‐mediated OIRD with fluoxetine and mimicking of OIRD with a TREK‐specific activator (BL 1249). Our results thus support a two‐tiered mechanism for OIRD: Primary suppression of network‐driven rhythmogenesis due to MOR1‐mediated reduction of excitatory synaptic transmission across the network, and a secondary role for intrinsic hyperpolarization driven by MOR1‐activated TREK K+ channels.Support or Funding InformationNIH R01HL126523, R01HL144801