New modification of the open field was designed to improve automation of the test. The main innovations were: (1) transmitted lighting and (2) estimation of probability to find pixels associated with an animal in the selected region of arena as an objective index of spatial preference. Transmitted (inverted) lighting significantly ameliorated the contrast between an animal and arena and allowed to track white animals with similar efficacy as colored ones. Probability as a measure of preference of selected region was mathematically proved and experimentally verified. A good correlation between probability and classic indices of spatial preference (number of region entries and time spent therein) was shown. The algorithm of calculation of probability to find pixels associated with an animal in the selected region was implemented in the EthoStudio software. Significant interstrain differences in locomotion and the central zone preference (index of anxiety) were shown using the inverted lighting and the EthoStudio software in mice of six inbred strains. The effects of arena shape (circle or square) and a novel object presence in the center of arena on the open field behavior in mice were studied.