CRISPR-Cas9 is quickly becoming a standard genome editing tool and has been used in various model organisms. It has yet to be implemented or optimized for use in Tetrahymena or ciliates in general despite the implications it has in facilitating germline modifications in binucleate systems. Cas9 has been shown to localize to the nucleus and gain access to DNA in other organisms without any nuclear localization sequence modifications. While this is standard in most systems, the presence of both a transcriptionally active macronucleus and a transcriptionally silent, germinal micronucleus, which has very different membrane characteristics, presents a unique question for ciliates: is Cas9 targeted to one or both nuclei? To answer this question we transformed Tetrahymena thermophila with an expression cassette, provided by Dr. Douglas Chalker and Dr. Robert Coyne, containing Cas9 fused to a micronucleus specific linker histone (MLH) and YFP under the inducible MTT1 promoter. We looked at Cas9 expression in induced transgenic cells during vegetative growth, starvation, early conjugation, and late conjugation. No observable signal was seen in the micronucleus or the macronucleus using either YFP fluorescence or anti-Cas9 antibody immunofluorescence, although some punctate staining was observed in the cytoplasm. Western blots of total cell protein extracted from induced cells probed with anti-Cas9 antibodies also showed no detectable signal corresponding to Cas9. Based on these observations, we hypothesized that the very large fusion protein containing Cas9, MLH, and YFP might be unstable or unable to enter the nuclei and may be rapidly degraded by the cell. To test this hypothesis we constructed two vectors, one containing just Cas9 fused only to YFP and one containing just Cas9. Using these smaller constructs, we intend to determine if Cas9 successfully enters either the micronucleus or the macronucleus during vegetative growth or conjugation. This work will help to establish and optimize conditions for Cas9 localization in Tetrahymena, and is an important first step in implementing CRISPR technology in this system.