16-3 is a temperate phage of the symbiotic nitrogen-fixing bacterium Rhizobium meliloti 41. Its prophage state and immunity against superinfection by homoimmune phages are governed by a complex set of controls: the immC and immX repressor systems and the avirT element are all located in well-separated, distinct regions which span 25 kb on the bacteriophage chromosome. The anatomy and function of the immC region are well documented; however, fewer analyses have addressed the immX and avirT regions. We focused in this paper on the immX region and dissected it into two major parts: X(U/L) and X(V). The X(U/L) part (0.6 kb) contained two overlapping cistrons, X(U) and X(L), coding for proteins pXU and pXL, respectively. Inactivation of either gene inactivated the repressor function of the immX region. Loss-of-function mutants of X(U) and X(L) complemented each other in trans in double lysogens. The X(V) part (1 kb) contained a target for X(U/L) repressor action. Mutations at three sites in X(V) led to various degree of ImmX insensitivity in a hierarchic manner. Two sites (X(V1) and X(V3)) exhibited the inverted-repeat structures characteristic of many repressor binding sites. However, X(V1) could also be folded into a transcription terminator. Of the two immunity regions of 16-3, immX seems to be unique both in its complex genetic anatomy and in its sequence. To date, no DNA or peptide sequence homologous to that of ImmX has been found in the data banks. In contrast, immC shares properties of a number of immunity systems commonly found in temperate phages.