Abstract: Behavior of open defecation is defecation in open areas (fields, gardens, rivers). The most important problem in the behavior of open defecation is actually a stool, as if the human who produced it suffered from the digestive tract, then the sludge may be contaminated with Escherichia coli (E. coli) and fecal streptococci which are often found in the human digestive tract. When E. coli is entered on disease transmission media such as food and drinking water, and enter in the digestive tract that are declining immunity would make a person is suffering from diarrhea. The research objective was to determine the relationship between knowledge and availability of toilet with behavior of open defecation. This research methods is correlational analytic with cross sectional approach. The population is community in Oesao Village East Kupang Sub-District, Camplong II Village Fatuleu Sub-District and Noelbaki Village Central Kupang Sub-District, by simple random sampling technique samples obtained 105 respondents. This study was conducted in January-February 2016. The results based on chi-square test showed p value 0,004 dan 0,019. The conclusion there is relationship between knowledge and availability of toilet with behavior of open defecation.