Primary cultures of renal proximal tubule cells (PTC) have been widely used to investigate tubule cell function. They provide a model system where confounding influences of renal haemodynamics, cell heterogeneity, and neural activity are eliminated. Additionally they are likely to more closely resemble PTC in vivo than established kidney cell lines, which are often virally immortalised and are of uncertain origin. This chapter describes a method used in our laboratories to isolate and culture pure populations of human PTC. The cortex is dissected away from the medulla and minced finely. Following collagenase digestion, the cells are passed through a sieve and separated on a Percoll density gradient. An almost pure population of tubule fragments form a band at the base of the gradient. Cultured in a hormonally defined serum-free growth media, they form a tightly packed monolayer that retains the differentiated characteristics of PTC for up to three passages.