Methylation and demethylation of the methyl-accepting chemotaxis protein (MCP) was studied in vitro. The in vitro MCP methylating system showed the following characteristics. 1. Multiple bands of methylated MCP were produced. 2. The reaction could be separated into two phases: the initial phase, in which the level of methylation increased as a result of multiple methylation; and the stationary phase, in which methylation and demethylation took place at the same velocity without apparent change in the level of methylation. 3. Pulse-chase analysis of the reaction showed that MCP was demethylated preferentially at the relatively highly methylated state. 4. The behavior of MCP in this system was similar to that of MCP stimulated in vivo with attractant. Based on the above results, an autonomous control mechanism of the level of MCP methylation is discussed.