Transient transfection is an excellent method for the expression and study of cell-surface, heteromeric ion channels. The cell type, the total amount of DNA, the combination of subunits and the ratio of subunit DNA are all important parameters to consider when attempting to optimize expression. A serious drawback of this method is that the efficiency of subunit assembly is very low in comparison to the efficiency of assembly for stably expressed heteromeric ion channels. The low efficiency of assembly prevents use of transient expression methods for detailed studies of heteromeric AChR assembly, and caution should be taken in the use of these methods for the study of intracellular heteromeric ion channel subunits. After the transient expression of heteromeric AChR subunits, virtually all of the expressing cells contained all four AChR subunits. However, the subunits were heterogeneously distributed among the cells, and the low efficiency of AChR assembly appears to be due to cell-to-cell variations in the ratio of the four subunits.