Human endogenous retrovirus K (HERV-K) is the most intact retrovirus in the human genome. There are multiple full-length or near full-length HERV-K proviruses in it. To analyze which HERV-K proviruses give rise to viral transcripts in cancer cell lines and to test whether ionizing radiation can alter the levels of HERV-K transcripts, RT-PCR studies were undertaken using multiple human cancer cell lines. Primers from several positions in the viral genome were used and included pairs designed to cross splice junctions in viral RNAs. In the absence of ionizing radiation, transcripts were detected from multiple HERV-K proviruses in cell lines from human prostate, cervical, head and neck, or breast cancers, and the proviruses from which the transcripts originated varied among the different lines. Only one of 13 cell lines tested (cervical cancer line C33A) failed to show HERV-K transcripts. Spliced RNAs detected included viral RNAs spliced as expected at the conventional viral splice sites, plus several alternatively spliced RNAs. Alternatively spliced transcripts arose from specific proviruses, and were detected in most of the cell lines used. Quantitative RT-PCR was performed to assess the effects of ionizing radiation. These analyses showed that HERV-K transcripts were elevated in four of twelve lines tested, specifically all three prostate cancer lines used and one breast cancer line. The increases were transient, peaking at 24 hours following a single dose of gamma-irradiation that ranged from 2.5 to 20 Gy, and returning to baseline levels by 72 hours. In summary, these studies showed that ionizing radiation can affect the levels of HERV-K transcripts in cells, and these effects vary among different cells. The changes in HERV-K transcript levels might affect multiple biological processes in cells, and future studies of the effects of ionizing radiation on HERV-K are worth pursuing.