Morphine and several other opioids are important drugs for the treatment of acute and chronic pain. Opioid-induced analgesia is predominantly mediated by the μ-opioid receptor (MOR). When administered to humans, complex metabolic pathways lead to generation of many metabolites, nine of which may be considered major metabolites. While the properties of the two main compounds, morphine-6-glucuronide and morphine-3-glucuronide, are well described, the activity of other morphine metabolites is largely unknown. Here we performed an extensive pharmacological characterization by comparing efficacies and potencies of morphine and its nine major metabolites for the two main signaling pathways engaged by the human MOR, which occur via G(i)-protein activation and β-arrestins, respectively. We used radioligand binding studies and FRET-based methods to monitor MOR-mediated G(i)-protein activation and β-arrestin recruitment in single intact 293T cells. This approach identified two major groups of morphine metabolites, which we classified into "strong" and "weak" receptor ligands. Strong partial agonists morphine, morphine-6-glucuronide, normorphine, morphine-6-sulfate, 6-acetylmorphine and 3-acetylmorphine showed efficacies in the nanomolar range, while the weak metabolites morphine-N-oxide, morphine-3-sulfate, morphine-3-glucuronide and pseudomorphine activated MOR pathways only in the micromolar range. Interestingly, three metabolites, normorphine, 6-acetylmorphine and morphine-6-glucuronide, had lower potencies for Gi-protein activation but higher potencies and efficacies for β-arrestin recruitment than morphine itself, suggesting that they are biased towards β-arrestin pathways.