Abstract Background The Plasmodium falciparum entomological inoculation rate ( Pf EIR) is a measure of exposure to infectious mosquitoes. It is usually interpreted as the number of P. falciparum infective bites received by an individual during a season or annually (a Pf EIR). In an area of perennial transmission, the accuracy, precision and seasonal distribution (i.e. , month by month) of a Pf EIR were investigated. Data were drawn from three sites in Uganda with differing levels of transmission where falciparum malaria is transmitted mainly by Anopheles gambiae s.l. Estimates of a Pf EIR derived from human-landing catches – the classic method for estimating biting rates – were compared with data from CDC light traps, and with catches of knock down and exit traps separately and combined. Methods Entomological surveillance was carried out over one year in 2011/12 in three settings: Jinja, a peri-urban area with low transmission; Kanungu, a rural area with moderate transmission; and Nagongera, Tororo District, a rural area with exceptionally high malaria transmission. Three sampling approaches were used from randomly selected houses with collections occurring once a month: human-landing collections (eight houses), CDC light traps (100 houses) and paired knock-down and exit traps each month (ten houses) for each setting. Up to 50 mosquitoes per month from each household were tested for sporozoites with P. falciparum by ELISA. Human biting rate (HBR) data were estimated month by month. P. falciparum Sporozoite rate ( Pf SR) for yearly and monthly data and confidence intervals were estimated using the binomial exact test. Monthly and yearly estimates of the HBR, the Pf SR, and the Pf EIR were estimated and compared. Results The estimated a Pf EIR values using human-landing catch data were 3.8 (95% Confidence Intervals, CI 0-11.4) for Jinja, 26.6 (95% CI 7.6-49.4) for Kanungu, and 125 (95% CI 72.2-183.0) for Tororo. In general, the monthly Pf EIR values showed strong seasonal signals with two peaks from May-June and October-December, although the precise timing of the peaks differed between sites. Estimated HBRs using human-landing catches were strongly correlated with those made using CDC light traps (r 2  = 0.67, p