At least one type of Rathke cleft cyst has unique MR findings, specifically, high intensity on T1-weighted images and iso- to low intensity on T2-weighted images relative to white matter. To clarify the influence of cyst content on MR images, we analyzed the cyst content by biomedical methods after surgical removal.We studied five patients diagnosed with Rathke cleft cyst, whose MR images showed high intensity on T1-weighted images and iso- to low intensity on T2-weighted images. After surgery, total protein and cholesterol levels were quantified, and correlations of protein and cholesterol content with T1 and T2 signal intensities were performed in vitro.All five cysts had very high concentrations of protein (11,700-26,600 mg/dl, mean 17,940 mg/dl) with nearly no cholesterol (at most 2.0 mg/dl). Along with increases in protein concentration in vitro, the signal intensity of T1-weighted images increased, while that of T2-weighted images decreased. In contrast, the cholesterol concentration sequence influenced the signal intensity of neither T1- nor T2-weighted images.The unique MR finding of Rathke cleft cysts--high signal intensity on T1-weighted images and low signal intensity on T2-weighted images--might depend mainly on protein concentration, not on cholesterol.