This study describes the pharmacokinetics of intravenously administered (i.v.) fentanyl citrate, and its primary metabolite norfentanyl in Holstein calves. Eight calves (58.6 ± 2.2 kg), aged 3–4 weeks, were administered fentanyl citrate at a single dose of 5.0 μg/kg i.v. Blood samples were collected from 0 to 24 hr. Plasma (nor)fentanyl concentrations were determined using liquid chromatography with mass spectrometry and a lower limit of quantification (LLOQ) of 0.03 ng/ml. To explore the effect of analytical performance on fentanyl parameter estimation, the noncompartmental pharmacokinetic analysis was then repeated with a hypothetical LLOQ value of 0.05 ng/ml. Terminal elimination half‐life was estimated at 12.7 and 3.6 hr for fentanyl and norfentanyl, respectively. For fentanyl, systemic clearance was estimated at 2.0 L hr−1 kg−1, volume of distribution at steady‐state was 24.8 L/kg and extraction ratio was 0.42. At a hypothetical LLOQ of 0.05 ng/ml fentanyl half‐life, volume of distribution at steady‐state and clearance were, respectively, of 3.0 hr, 8.8 L/kg and 3.4 L kg−1 hr−1. Fentanyl citrate administered i.v. at 5.0 μg/kg can reach levels associated with analgesia in other species. Pharmacokinetic parameters should be interpreted with respect to LLOQ, as lower limits can influence estimated parameters, such as elimination half‐life or systemic clearance and have significant impact on dosage regimen selection in clinical practice.