Calcium is a critical structural and regulatory nutrient in plants. However, mechanisms of its uptake by root cells are poorly understood. We have found that Ca2+ influx in Arabidopsis root epidermal protoplasts is mediated by voltage-independent rapidly activating Ca2+-permeable non-selective cation channels (NSCCs). NSCCs showed the following permeability (P) sequence: PCa (1.00) = PBa (0.93) > PZn (0.51), PCa/PNa = 0.19, PCa/PK = 0.14. They were inhibited by quinine, Gd3+, La3+ and the His modifier diethylpyrocarbonate, but not by the Ca2+ or K+ channel antagonists, verapamil and tetraethylammonium (TEA+). Single channel conductance measured in 20 mm external Ca2+ was 5.9 pS. Calcium-permeable NSCCs co-existed with hyperpolarisation-activated Ca2+ channels (HACCs), which activated 40–60 min after forming the whole-cell configuration. HACCs activated at voltages <−130 to −150 mV, showed slow activation kinetics and were regulated by cytosolic Ca2+ ([Ca2+]cyt). Using aequorin-expressing plants, a linear relationship between membrane potential (Vm) and resting [Ca2+]cyt was observed, indicating the involvement of NSCCs. Intact root 45Ca2+ influx was reduced by Gd3+ (NSCC blocker) but was verapamil and TEA+ insensitive. In the root elongation zone, both root net Ca2+ influx (measured by Ca2+-selective vibrating microelectrode) and NSCC activity were increased compared to the mature epidermis, suggesting the involvement of NSCC in growth. A Ca2+ acquisition system based on NSCC and HACC co-existence is proposed. In mature epidermal cells, NSCC-mediated Ca2+ influx dominates whereas in specialised root cells (root hairs and elongation zone cells) where elevated [Ca2+]cyt activates HACCs, HACC-mediated Ca2+ influx predominates.