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Background: We investigated the biting patterns and seasonal abundances of Anopheles gambiae s.l. and An. funestus mosquitoes in Kamuli District, Uganda. Methods: Hourly indoor and outdoor catches of human biting mosquitoes were sampled from 19.00 to 07.00 hours for four consecutive nights each month using bed net traps in forty-eight houses randomly selected from Bugabula county where insecticide-treated bed nets (ITNs) had been used for at least five years and Budiope county where ITNs had not been used. The indoor and outdoor human-biting fractions, time of biting of the anophelines and climatic data were recorded from January to December 2010. Data were analysed using Multi-way analysis of variance, Kruskal-wallis rank sum test and Pearson correlation. The number of mosquitoes caught biting humans and resting indoors, the indoor and outdoor human biting densities and biting rates during different hours of the night, and mosquito abundances for a twelve-month sampling period in both zones are reported. Results: Approximately four times more Anopheles mosquitoes were caught biting humans in Budiope County than in the Bugabula zone, with An. gambiae s.l. catches exceeding those of An funestus. In both zones, peak night biting occurred between 23.00 and 05.00 hours. The majority of bites occurred between 03.00 and 06.00 hours for both Anopheles gambiae s.l. and funestus group. Outdoor biting densities of Anopheles gambiae s.l. exceeded the indoor biting densities throughout the night in both zones, while the indoor and outdoor human biting densities of An. funestus group were apparently equal. The outdoor and indoor human biting rates were similar in both zones. In Bugabula county, the abundance of An. gambiae s.l. was rainfall-dependent, while the An. funestus group could thrive with or without rain fall. In Budiope county, both An. gambiae s.l. and An. funestus mosquitoes thrived all year round regardless of the amount of rainfall. Conclusion: Considering the biting patterns, and seasonal abundances exhibited by Anopheles gambiae s.l. and An. funestus mosquitoes in Kamuli district, intensive use of ITNs combined with indoor residual spraying, environmental management and improved house designs in the context of integrated vector management may be the appropriate vector control strategy