Properties of particle-bound neuraminidase activity of ratkidney were studied using a colorimetric assay for measuring released free sialic acid; several glycoproteins were used as substrates. Neuraminidase specific activities of cortex and medulla were similar, but lower activity was obtained with glomerular preparations. The nonionic detergent Triton X-100 solubilized the neuraminidase but had a moderately inhibitory effect on its activity. Substrate competition studies indicated the presence of only one enzyme in rat kidney. Tritium sialic acid-labeled fetuin and Tamm-Horsfall glycoprotein were used as substrates in a radioactive assay for kidney neuraminidase. This assay system permits detection of low levels of activity when combined with a chromatographic procedure for separating free sialic acid.