Cerebrospinal fluid (CSF) is secreted by the choroid plexus, and has a key role in brain homeostasis and preservation of normal neural function (Davson & Segal, 1996). It is one of the key tissues that is useful in the search for biomarkers of neurodegenerative disorders. CSF contains a high salt (>150 mmol/l) and lower protein concentration (approximate 0.4 mg/ml) than plasma (approximate 55 mg/ml). It needs to be de-salted and concentrated before two-dimensional (2-D) electrophoresis (Yuan et al. 2002). In this study, several CSF preparation methods were compared using a same ovine CSF sample. Three adult Clun Forest sheep were anaesthetized with i.v. thiopentone sodium (20 mg kg^-1). CSF samples were collected from cisterna magna by a needle puncture. An aliquot of CSF (250 μl, approximate 100 μg protein) was prepared using 4 methods: 1. Acetone precipitation; 2. 2-D clean-up kit (Amersham); 3. Vivaspin 0.5 ml concentrator (Sigma); 4. Bio-rad spin column (Hercules, CA, USA). CSF proteins in 400 μl rehydration buffer were re-swelled onto 18 cm non-linear DryStrips and focused with an IPGphor unit. After equilibration, each strip was loaded onto 12% acrylamide gel, and the proteins were subjected to electrophoresis with a ISO-DALT cell. The gel was then silver stained and scanned. The image was analyzed using Phorex software. CSF from the same sheep was treated with 4 different methods before 2-D electrophoresis. CSF preparations with precipitation (acetone precipitation and 2-D clean-up kit) produced more spots than the Sigma and Bio-Rad spin columns (Table 1, Fig. 1). One gel with 2-D clean-up kit preparation was then used as the master gel. The gels with 2-D clean-up kit preparation matched 88(8)% of the master gel (Table 1). Using the other 3 methods, a large and high density spot in the middle Mr region appeared on the gels (Fig. 1). CSF preparation by precipitation preserves more proteins than the spin column methods. The large spot missing from 2-D clean-up kit preparation needs to be identified.