Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.