Abstract The carcinogen β-propiolactone (BPL) was reacted with thymidine-5′-monophosphoric acid (dThd5′P) at pH 1.8 and various temperatures in order to determine whether BPL forms phosphodiesters with dThd5′P. The acidic conditions were chosen in an attempt to avoid complicating ring alkylations. Paper chromatographic analyses of the reaction products revealed the presence of unreacted dThd5′P and a second ultraviolet absorbing compound designated Compound F2. F2 and dThd5′P had identical UV spectra at pH 1 and 13. The major component of the reaction mixture was unreacted dThd5′P. F2 represented 10% of the ultraviolet absorbing compounds extracted from paper chromatograms following reactions at 37, 50, 63 and 76°C and 5% at 24°C. On the basis of ultraviolet spectra, treatment with snake venom phosphodiesterase and alkaline phosphatase (each at pH 7.2 and 9.0), labeling experiments with β-[ 14C]propiolactone and hydrolyses at pH 1, 7, 9, 11 and 13 at 37°C for 18 h (no detectable effects) the new phosphodiester, 5′-O-(2-carboxyethyl)phosphonothymidine was assigned to F2. When BPL was reacted with dThd5′P at pH 6.6–7.0 at 37°C, 2 new UV absorbing compounds were observed on paper chromatograms in addition to dThd5′P and F2, and were designated Compounds F3 and F4. UV spectra of F3 and F4 were identical to dThd5′P at pH 1 and 13. dThd5′P represented 36% of the ultraviolet absorbing compounds extracted from paper chromatograms, the remainder being F2 (36%), F3 (24%) and F4 (4%). Treatment of F3 with snake venom phosphodiesterase at both pH 7.2 and 9.0 resulted in complete conversion to dThd5′P. F3 is thus a phosphodiester. Hydrolysis of F3 at pH 13 and 11 (37°C and 18 h) resulted in complete conversion of F3 to F2 and a trace amount of dThd5′P while F3 was unreactive at pH 9, 7 and 1. 3′-O-Acetylthymidine was completely hydrolyzed to thymidine at pH 13, 11 and 9 and partially (65%) hydrolyzed at pH 1. A 3′-derivative of dThd5′P (ether or ester) was thus ruled out as a structure for F3. After considering a number of structures, a tentative assignment of a structure containing a 6-membered ring phosphate ester-acyl phosphate derivative was assigned to F3 based on the evidence presented and a positive acyl phosphate test for F3. When the reaction between BPL and dThd5′P was conducted at pH 7.0–7.5, 7.5–8.0 and 8.0–8.5 the results were similar to the reaction conducted at pH 6.6–7.0. No ring alkylation was detected. The significance of these findings in terms of possible effects of BPL on messenger RNA activity, excision-repair of DNA, DNA synthesis and possible alterations in gene expression following reaction of BPL with phosphorylated histones and nonhistone chromosomal proteins is discussed.