In recent years major progress has been achieved in the understanding of transport processes in higher plants. The boom in the field of molecular plant physiology led to the identification and characterization of membrane transporters with transport activities for potassium, calcium, sugars, nitrate, ammonium, sulphate, phosphate, amino acids, peptides, and metal ions. Such progress was hardly feasible without heterologous expression of the isolated transport proteins. This review summarizes the different approaches in characterizing plant membrane transporters using heterologous expression systems. By presenting concrete examples, it outlines different cloning strategies, displays the methods used for (i) expression of transport proteins and detection of their function, (ii) biochemical analyses, (iii) explorations of the structure-function relationship through mutational analysis, and concludes with a discussion about the physiological relevance of the analyses in heterologous expression systems.