Objective: To study 1) whether the accuracy of point-of-care (POC) urine tests (dipstick, phase-contrast microscopy and culture) differs when performed on first-void urine (FVU) compared to mid-stream urine (MSU), and 2) if a delay of analysis up to four hours affects the accuracy of POC tests when performed on urine from symptomatic of urinary tract infection (UTI), non-pregnant women in general practice. Design: Prospective diagnostic study using paired samples. Setting/Intervention: Three general practices in Copenhagen. Each woman delivered FVU and MSU samples from the same void. As a reference standard, 8 ml of MSU was sent for culture at the Microbiology Department. Patients: 117 women with one or more symptoms of UTI. Main outcome measures: Sensitivity, specificity and agreement with the reference standard of FVU and MSU with different time delays (zero vs. one vs. four hours) as compared to reference standard (MSU at time zero in boric acid tubes). Results: All three POC tests performed on MSU were significantly more in agreement with the reference than when performed on FVU when analysis was done immediately. The error rate was 16% for MSU vs. 23% for FVU with POC culture, 27% vs. 40% with microscopy and 25% vs. 33% with dipstick testing. Delay of analysis up to four hours did not decrease agreement with the reference. Conclusion/Implication: MSU samples should be used in general practice for optimal accuracy of POC tests. Analysis can be delayed up to four hours.Key points Point-of-care tests (dipstick testing, microscopy and culture) for diagnosing urinary tract infection performed on mid-stream urine samples are significantly more accurate than when performed on first-void urine samples. Delay of analysis up to four hours did not decrease the accuracy of any of the point-of-care tests. Point-of-care culture was more accurate than dipstick and microscopy both when performed on mid-stream urine and first-void urine The main contaminant in first-void urine samples was Enterococci spp., which contributed to the majority of false positives.