Intrathecal administration is an important route for drug delivery, and in pharmacology and toxicology studies, cerebrospinal fluid (CSF) collection and analysis is required for evaluating blood-brain barrier penetration and central nervous system exposure. The characteristics of CSF in commonly used nonrodent models are lacking. The purpose of this study is to evaluate and provide some insights into normal cellular and biochemical composition of CSF as well as diffusion potential following intrathecal injection across several nonrodent species. Cerebrospinal fluid samples were collected from the cerebellomedullary cistern of beagle dogs, cynomolgus monkeys, and Göttingen minipigs and analyzed for clinical chemistry and cytological evaluation. Diffusion into the intrathecal space following intrathecal injection was assessed following administration of a contrast agent using fluoroscopy. The predominant cell types identified in CSF samples were lymphocytes and monocytoid cells; however, lymphocytes were represented in a higher percentage in dogs and monkeys as opposed to monocytoid cells in minipigs. Clinical chemistry parameters in CSF revealed higher Cl- concentrations than plasma, but lower K+, Ca2+, phosphorus, glucose, creatinine, and total protein levels consistent across all 3 species. Diffusion rates following intrathecal injection of iodixanol showed some variability with dogs, showing the greatest diffusion distance; however, the longest diffusion time through the intervertebral space, followed by monkeys and minipigs. Minimal diffusion was observed in minipigs, which could have been attributed to anatomical spinal constraints that have been previously identified in this species.