The purpose of this work was to study the development of astroglial cells in patterned neuronal cultures. Hippocampal neurons, derived from embryonic stage (E18) rats and cultured in serum-free Neurobasal/B27 medium, grew to follow patterns of poly(D-lysine) created by micro-contact printing. The growth of the astroglial cells and the co-localization of neurons and astroglial cells were measured for up to one month using fluorescence immunostaining of neurons and astroglial cells. Neurons grew to form square patterns within 2 weeks, while astroglia only started to emerge in the same period. Astroglial cells continued to proliferate for a month following a general growth curve. Over 90% of the astroglial cell area co-localized with neurons (within 2 mum) at an early stage of astroglial development (13 DIV). Over the remaining period, astroglial cells proliferated and the co-localization was 80%. Hence, in these culture conditions astroglial cells develop 2-3 weeks later than neurons but remain highly co-localized with neurons.