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The dopaminergic system has been suggested to serve as a functional target for ketamine, especially with its addictive and antidepressant effects. In this work, intracellular vesicle impact electrochemical cytometry and single cell amperometry were applied to a model cell line to show that the intravesicular contents and number of exocytotic events increased and the fraction of release decreased after treatment by subanesthetic ketamine (1 μM for 15 min). In addition, time‐of‐flight secondary ion mass spectrometry was used to show that cellular lipids change following acute ketamine administration relative to control cells with an increased proportion of low‐curvature lipids and a decreased proportion of high‐curvature lipids. We propose that ketamine modulates dopaminergic function with a dose‐dependent pharmacologic action by dominantly interacting with different proteins and changing lipid composition. It might be helpful to better understand the relationship of the dopaminergic system and drug abuse, as well as anesthetic and antidepressant effects. [ABSTRACT FROM AUTHOR]