The objective of this study was to develop an analytical methodology for the determination of metomyl in soils through the inhibition of the enzime acetylcolinesterase (AChE), also regarding the influence of soil fertility parameters. The soil samples were collected from agricultural areas in the microbasin of São Lourenço stream, in Nova Friburgo district, RJ, in order to contemplate different soil categories and the areas agricultural management. This area is one of the main olericole producers in Rio de Janeiro state, with records of intensive use of agrotoxics. A research on the types of cultures and agrotoxic use and their respective application methods was conducted, through interviews with laborers, producers and suppliers, in order to acquaint ourselves with the area. The extraction and enzymatic determination methods were optimized, indicating no need for clean-up, which renders the procedure an easy operation, with smaller analysis periods and reagent use. The use of microplates in the measurement of the enzymatic activity of AChE permitted the use of smaller amounts of enzyme and substrate (Acetilcoline), reducing the analysis periods. Comparisons between recently contaminated samples and those stocked for periods varying from 24h to 1 week, in different temperature conditions (25ºC, 4ºC and -15ºC), indicated that a higher sample stability and conservation can be obtained at 4ºC, although this is still dependent on the concentration of this methylcarbamate and the matrix constituents. The optimized methodology was applied in the quantification of methomyl in soil samples of the area successfully, indicating that it can be used in environmental evaluation diagnosis and in anticollinergic pesticides (carbamate and/or organophosphates) monitoring programs. The availability of practical and inexpensive methodologies helps controlling government organs in the effective implementation of routine analysis s that aim to subsidiate government decisiontakings and the elaboration of certificates for phyto-sanitary control.