Recent estimates indicate that approximately 60% of human genes include alternative polyadenylation sites. Hence, control of alternative polyadenylation can have a great impact on gene expression and cellular function. Cleavage factor (CF) Im is a 3'-end processing factor that is essential for in vitro processing. CFIm purified from HeLa cells is associated with three polypeptides of 25, 59 and 68 kD, and it is generally thought to be a heterodimer composed of the 25-kD subunit and one of the larger subunits. Previously, we serendipitously discovered that knockdown of CFIm25 causes an upstream shift in the utilization of alternative polyadenylation sites. Here, we investigated whether this is because of an inherent property of the CFIm complex and, if so, what structural elements are important for its function. The major conclusions of this study are that (i) contrary to previous assumptions, CFIm forms stable heterotetramers through dimerization of CFIm25 and (ii) the CFIm complex per se is responsible for the control of alternative polyadenylation. (iii) However, the structurally related CFIm68 and CFIm59 are functionally redundant and (iv) CFIm68 appears to have a higher specific activity. Thus, this study establishes that CFIm not only plays a general role in 3'-end processing but also plays a regulatory role in poly(A) site selection.