An enzyme-linked antibody centrifuge assay for the detection of Escherichia coli pilus K99 was developed and shown to be a specific and quantitative assay for the detection of cell-bound K99. The data presented demonstrate the usefulness of the assay as a diagnostic tool. Using the assay, several factors that affect expression of K99 were investigated. Expression of K99 was dependent upon the degree of aeration provided: nonaerated bacteria produced little or no K99, whereas aerated bacteria produced large amounts of K99. K99 also appeared to be produced only by logarithmically growing cells, whereas there was a demonstrable decline in the amount of K99 per cell during stationary phase. Glucose was shown to repress K99 expression. At 0.5% glucose, K99 expression was highly repressed. Glucose-mediated repression could be overcome by the addition of cyclic adenosine 3',5'-monophosphate. Several other carbon sources also inhibited K99 expression, including pyruvate, arabinose, and lactose; glycerol was stimulatory.