Loss of serum-free L-cell colony stimulating factor (CSF) activity was noted when non-sterile or ethylene oxide sterilized Millipore filters were employed; autoclaved membranes showed no inactivation. The reduction in CSF did not appear to be due to release of inhibitory or inactivating substances as extensive rinsing with various solvents did not prevent loss of CSF activity. In addition, these filtrates did not inhibit the activity of standard CSF. Less pure sources of CSF, including standard serum containing L-cell CSF and human urinary concentrate, did not lose activity upon filtration. Moreover, the addition of bovine serum albumin to serum-free CSF completely prevented membrane filtration loss. The CSF loss could be prevented by treatment of the membranes with buffers contraining 0.05% polyethylene glycol (PEG). These findings show that Millipore membranes can bind significant quantities of partially purified CSF. It is important in purification studies to recognize that CSF loss can be prevented by addition of PEG to all buffer systems.