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The objective of this study was to explore knowledge of, attitudes towards and practice of post-exposure prophylaxis (PEP) among healthcare workers (HCWs) in the Thika district, Kenya. We used site and population-based surveys, qualitative interviews and operational research with 650 staff at risk of needlestick injuries (NSIs). Research was conducted over a 5-year period in five phases: (1) a bio-safety assessment; (2) a staff survey: serum drawn for anonymous HIV testing; (3) interventions: biosafety measures, antiretrovirals for PEP and hepatitis B vaccine; (4) a repeat survey to assess uptake and acceptability of interventions; in-depth group and individual interviews were conducted; and (5) health system monitoring outside a research setting. The main outcome measures were bio-safety standards in clinical areas, knowledge, attitudes and practice as regards to PEP, HIV-sero-prevalence in healthcare workers, uptake of interventions, reasons for poor uptake elucidated and sustainability indicators. Results showed that HCWs had the same HIV sero-prevalence as the general population but were at risk from poor bio-safety. The incidence of NSIs was 0.97 per healthcare worker per year. Twenty-one percent had had an HIV test in the last year. After one year there was a significant drop in the number of NSIs (OR: 0.4; CI: 0.3-0.6; p<0.001) and a significant increase in the number of HCWs accessing HIV testing (OR: 1.55; CI: 1.2-2.1; p=0.003). In comparison to uptake of hepatitis B vaccination (88% of those requiring vaccine) the uptake of PEP was low (4% of those who had NSIs). In-depth interviews revealed this was due to HCWs fear of HIV testing and their perception of NSIs as low risk. We concluded that Bio-safety remains the most significant intervention through reducing the number of NSIs. Post-exposure prophylaxis can be made readily available in a Kenyan district. However, where HIV testing remains stigmatised uptake will be limited - particularly in the initial phases of a programme.