Three studies were conducted to investigate the reasons for the loss of countable organisms in the Information Collection Rule (ICR) protozoan method. In the first study, 35 laboratories, representing 61 principal analysts (PAs) who participated in an initial round of laboratory approvals, and who had completed their first set of protozoan performance evaluation (PE) samples, received quality control vials containing known concentrations of Giardia cysts and Cryptosporidium oocysts. The ICR method was used and the recovery of cysts and oocysts was calculated and compared to PE approval results. The second study was performed by the reference laboratory to characterize losses of oocysts through the Hoefer manifold in the ICR method. In the third study, a suspension stained vial, that had been counted using a combined fluorescence microscopy and hemacytometer chamber counting technique, was employed to evaluate where Giardia cysts and Cryptosporidium oocysts were lost on the manifold. This paper presents the results of these three studies.