Background <p>In vitro evaluation of substances utilizing the putative proton‑coupled organic cation (H⁺/OC) antiporter for active uptake across the blood-brain barrier (BBB) and brain cell membranes requires a thorough understanding of cellular pharmacokinetics supported by reliable translational readouts. This study assessed the rate and extent of uptake of the antiporter substrate oxycodone in brain endothelial and parenchymal cells at clinically relevant concentrations, exploring the suitability of various cell models for investigating active drug transport.</p> Methods <p>Transcellular transport studies were performed using primary brain endothelial cells (BECs) from pig, rat, and mouse, alongside uptake assays in immortalized human cerebral microvascular endothelial cells (hCMEC/D3) and rat brain slices. Drug uptake was estimated by combining transport data with non-specific binding data via equilibrium dialysis. The effect of interleukin-6 (IL-6) on oxycodone uptake was tested in hCMEC/D3 cells. The unbound intracellular-to-extracellular concentration ratio (K_p,uu,cell) and efflux ratio were used to compare the extent of net uptake across models and evaluate the presence of active uptake.</p> Results <p>Based on cellular pharmacokinetic parameters, both primary BECs and hCMEC/D3 demonstrated active uptake of oxycodone. Mean permeability across primary BECs ranged between 0.9 × 10⁻⁵ (pig) and 1.8 × 10⁻⁵ (rat) cm/s. Transport extent, reflected by 1/Efflux Ratio values of 1.5 (pig) and 2.4 (rat), aligned with in vivo unbound brain-to-plasma concentration ratios, K_p,uu,brain, indicating predominant active uptake. In hCMEC/D3 cells, the uptake rate was time- and concentration-dependent within 60&#xa0;min and 10–5000 nM concentration range, while the uptake extent, assessed by K_p,uu,cell, was 1.7 and independent of both. IL-6 increased the extent of uptake to 148% of control, without affecting the rate. The extent of uptake into parenchymal cells was also concentration-independent, with K_p,uu,cell values around 1.8, similar to endothelial cells.</p> Conclusions <p>These findings provide insight into oxycodone distribution across the BBB and into brain parenchymal cells, particularly emphasizing the contribution of probable active transport mechanisms at multiple barriers. The results highlight the importance of assessing both the rate and extent of transport and support the utility of K_p,uu,cell as a key metric for comparing drug transport across models. Both primary BECs and hCMEC/D3 cells are reliable tools for assessing substrate uptake in drug development, particularly for H⁺/OC antiporter substrates, and hold translational potential for mechanistic studies.</p> Graphical Abstract <p></p>