Background: A system for the preparation of sterilized instruments with unidirectional horizontal air flow (UDHF) has several advantages over a unidirectional down flow system (UDDF). The advantages are based on the installation of the system being more flexible and easier to use, no cooling of the air flow being necessary and less air being needed for circulation, resulting in reduced energy use. Objectives: The objective of this study was to determine whether a system with UDHF performs equal or superior to a system with UDDF in terms of prevention of contamination of the air (the presence of particles and micro-organisms) during the laying-up process. Methods: The degree of protection (DP) offered by two UDHF system variants and two UDDF system variants was determined for several static set-ups and a dynamic simulation of the process. In addition to determining the level of protection for several categories of particle size, colony-forming units (CFU) were also measured during process simulations. Findings: When maximum protection (no particles present) is considered, the UDHF systems performed significantly better than the UDDF systems for particles >= 2.5 mu m. When particles were present, there was no significant difference between systems for particles >= 0.3 and >= 0.5 mu m. However, the performance of the UDHF system was superior to that of the UDDF system (DP) for particles >= 1.0 mu m representing the bacteria-carrying particles. During the process measurements, no CFU were found Conclusions: The UDHF system offers equal or superior protection to the UDDF system against contamination of the clean area within which the laying up takes place. Despite our finding that the differences did not always reach statistical significance (due to low background concentrations), there is a clear trend, from the small-sized particles (>= 1.0 mu m) up to the largest sizes considered, including bacteria-carrying particles, that demonstrates the superiority of the horizontal flow system.