OBJECTIVES: To establish the utility of a novel in-house method of CSF analysis using sedimentation cytology direct from the spinal needle for the detection of laboratory-defined pleocytosis. MATERIALS AND METHODS: In dogs and cats undergoing routine CSF analysis for investigation of neurological signs, an additional preparation was made at the patient's side by everting the spinal needle on a slide and sedimenting for at least 1 hour. Nucleated cellularity and differential counts were assessed and compared with "gold-standard" analysis. Variability of cell counts between observers and within slides using the new method was evaluated to optimise the procedure. RESULTS: Using a ×50 objective, at least 10 fields and an average of more than five cells per field were considered appropriate guidelines to achieve correct classification of samples (normal or pleocytosis). The new method had high sensitivity (89%) and specificity (100%) for the detection of laboratory-defined pleocytosis. Agreement on the type of pleocytosis was good. CLINICAL SIGNIFICANCE: Clinically useful information can be obtained from CSF samples in a patient-side setting without additional equipment. This technique may be of benefit if little fluid is available or if logistical constraints limit the availability of rapid specialist results.