Microfluidics can be used to generate flow-driven gradients of chemotropic guidance cues with precisely controlled steepnesses for indefinite lengths of time. Neuronal cells grown in the presence of these gradients can be studied for their response to the effects exerted by the cues. Here we describe a polydimethylsiloxane (PDMS) microfluidics chamber capable of producing linear gradients of soluble factors, stable for at least 18 h, suitable for axon guidance studies. Using this device we demonstrate turning of superior cervical ganglion axons by gradients of nerve growth factor (NGF). The chamber produces robust gradients, is inexpensive to mass produce, can be mounted on a tissue culture dish or glass coverslip for long term time-lapse microscopy imaging, and is suitable for immunostaining.